Spinosyn derivatives as insecticides

ABSTRACT

Compositions including derivatives of spinosyns and methods for the production of derivatives of spinosyns are provided. The spinosyn derivatives described herein include spinosyn derivatives functionalized on the C5-C6 double bond of spinosyn A to provide an additional ring system. The method produces spinosyn derivatives that exhibit activity towards insects, arachnids, and/or nematodes and are useful in the agricultural and animal health markets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase of International ApplicationNo. PCT/US2016/050034 filed on Sep. 2, 2016, and published on Mar. 9,2017 as International Publication No. WO 2017/040882 A1, whichapplication claims priority to and the benefit of U.S. ProvisionalPatent Application No. 62/214,083 filed Sep. 3, 2015; U.S. ProvisionalPatent Application No. 62/290,676 filed Feb. 3, 2016; U.S. ProvisionalPatent Application No. 62/303,015 filed Mar. 3, 2016; U.S. ProvisionalPatent Application No. 62/303,078 filed Mar. 3, 2016; and U.S.Provisional Patent Application No. 62/380,664 filed Aug. 29, 2016, thecontents of all of which are incorporated herein by reference in theirentireties.

FIELD

Described herein are spinosyn derivatives for use as agrichemicals.

BACKGROUND

Spinosyn refers to a large family of compounds produced from thefermentation of soil actinomycetes species of Saccharopolyspora. Theindividual components from the fermentation broth were subsequentlygiven the generic name of spinosyn to connect these compounds with theirproducing microorganism, Saccharopolyspora spinosa. Members of thespinosyn family share a core structure having a polyketide-derivedtetracyclic macrolide appended with two saccharides. There are manynaturally occurring variants, which exhibit potent insecticidalactivities against many commercially significant species that causeextensive damage to crops and other plants. Some of these variants alsoexhibit activity against important external parasites of livestock,companion animals and humans.

Fermentation of S. spinosa produces a natural mixture containingspinosyn A as the major component and spinosyn D as the minor componentand named spinosad. The structure of spinosyn A was determined by NMR,MS, and X-ray analyses and comprises a tetracyclic polyketide aglyconeto which is attached a neutral saccharide substituent(2,3,4-tri-O-methyl-α-L-rhamnosyl) on the C-9 hydroxyl group and anaminosugar moiety (β-D-forosaminyl) on the C-17 hydroxyl group. Thisspinosyn tetracyclic ring system composed of acis-anti-trans-5,6,5-tricyclic moiety fused to a 12-membered lactone isa unique ring system.

The second most abundant fermentation component is spinosyn D, which is6-methyl-spinosyn A. Spinosyn D is likely formed by incorporation ofpropionate instead of acetate at the appropriate stage during polyketideassembly.

Numerous structurally related compounds from various spinosynfermentations have now been isolated and identified. Their structuresfall into several general categories of single-type changes in theaglycone or saccharides of spinosyn A.

Spinosyns have a unique mechanism of action (MOA) involving disruptionof nicotinic acetylcholine receptors. When compared with many otherinsecticides, spinosyns generally show greater selectivity toward targetinsects and lesser activity against many beneficial predators.Structure-activity relationships (SARs) have been extensively studied,leading to development of a semisynthetic second-generation derivative,spinetoram (Kirst (2010)J. Antibiotics 63:101-111).

Studies to date have concluded that the mechanism(s) by which spinosynexerts its insecticidal action is different from those of any otherknown agents, and thus cross-resistance between spinosyn and otheragents was initially absent or low. However, as well known for otherinsecticides, continued usage is likely to exert selective pressures oninsects and to eventually provoke resistance.

The unique and highly complex core structure of the spinosyns hasprovided challenging opportunities for synthesis. Additionally, with theincrease of insect resistance, new spinosyn compounds and methods fortheir synthesis are needed.

SUMMARY

Spinosyn compounds and methods for making and using the spinosyncompounds are provided. The spinosyn compounds described herein exhibitactivity towards insects, arachnids, and nematodes and/or are useful inthe agricultural and animal health markets. The spinosyn compoundsdescribed herein exhibit activity comparable to or greater than thespinosyn-type natural products, often with an improved resistanceprofile over the natural products.

A spinosyn compound as described herein includes a compound of thefollowing formula:

or a salt thereof, wherein

is a single bond or a double bond; A is hydrogen or is selected from thegroup consisting of substituted or unsubstituted carbonyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl; B is selected from the group consisting ofsubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl; C is O or NH; R¹ is substituted orunsubstituted alkyl or substituted or unsubstituted aryl; X¹, X², and X³are each independently selected from O, S, N, NR, CR, and CR₂, whereineach R is independently selected from hydrogen, hydroxyl, substituted orunsubstituted amino, substituted or unsubstituted thio, substituted orunsubstituted alkoxy, substituted or unsubstituted aryloxy, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl,wherein when X¹ is O, X³ is not N. Optionally R¹ is C₁-C₆ alkyl or C₁-C₆aryl. Optionally, R¹ is ethyl.

Optionally, when X¹ and X² are selected from NR, CR, and CR₂, the Rgroups of X¹ and X² can combine to form a substituted or unsubstitutedcycloalkyl, substituted or unsubstituted cycloalkenyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstitutedheterocycloalkenyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl. Optionally, when X² and X³ are selectedfrom NR, CR, and CR₂, the R groups of X² and X³ can combine to form asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl,and substituted or unsubstituted heteroaryl.

Optionally, A comprises hydrogen, forosamine, or a forosaminederivative, such as a [(2R,5S,6R)-6-methyl-5-(methylamino)oxan-2-yl]oxygroup. Optionally, B comprises rhamnose or a rhamnose derivative, suchas a [(2R,5S)-3,4,5-dimethoxy-6-methyloxan-2-yl]oxy group(3,4,5-trimethoxyrhamnose) or a[(2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy group(4-ethoxy-3,5-dimethoxyrhamnose).

In some examples, A is forosamine; B is 3,4,5-trimethoxyrhamnose or4-ethoxy-3,5-dimethoxyrhamnose; C is O; R¹ is ethyl; X¹ is N, S, or NH;X² is CR; X³ is S, NH, N(CH₃), or N; and R is selected from hydrogen,substituted and unsubstituted alkyl (e.g., methyl, ethyl, isopropyl,tertbutyl, or halogenated alkyl), substituted and unsubstituted amino(e.g., phenylamino, alkylphenylamino, alkoxyphenylamino, halogenatedphenylamino, (benxyloxy)phenylamino, benzylamino, or benzoylamino),substituted and unsubstituted cycloalkyl (e.g., cyclopropyl),substituted or unsubstituted cycloalkenyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl,substituted and unsubstituted aryl (e.g., phenyl, halogenated phenyl,hydroxyphenyl, alkoxyphenyl, alkylphenyl, or halogenated alkylphenyl),substituted and unsubstituted heteroaryl (e.g. thiophenyl or pyridinyl),and substituted and unsubstituted thiol (e.g. alkylthiol).

In some examples, A is a forosamine derivative; B is(2R,5S)-3,4,5-dimethoxy-6-methyloxy-; C is O; R¹ is ethyl; X¹ is N; X²is CR; X³ is S; and R is selected from substituted and unsubstitutedalkyl (e.g., methyl, ethyl, isopropyl, tertbutyl, or halogenated alkyl)and substituted and unsubstituted aryl (e.g., phenyl, halogenatedphenyl, hydroxyphenyl, alkoxyphenyl, alkylphenyl, or halogenatedalkylphenyl). In some examples, one of the methyl groups on the nitrogenof the forosamine derivative is substituted with a group selected fromhydrogen, tosyl, benzyl, propyl, 5-methylfuran-2-ly, chlorofluorobenzyl,ethyl, cyclopropyl, octonoyl, (2-methyl)propanoyl, benzoyl, propenoyl,or a —S(CH₃)O₂ group.

In some examples A is hydrogen; B is(2R,5S)-3,4,5-dimethoxy-6-methyloxy-; C is O; R¹ is ethyl; X¹ is N; X²is CR; X³ is S; and R is selected from substituted and unsubstitutedalkyl (e.g., methyl, ethyl, isopropyl, tertbutyl, or halogenated alkyl)and substituted and unsubstituted aryl (e.g., phenyl, halogenatedphenyl, hydroxyphenyl, alkoxyphenyl, alkylphenyl, or halogenatedalkylphenyl).

In some examples, A is forosamine; B is(2R,5S)-3,4,5-dimethoxy-6-methyloxy-; C is O; R¹ is ethyl; X¹ is N; X²is CR; X³ is NR; and the R groups of X² and X³ combine to form asubstituted or unsubstituted heteroaryl ring (e.g. a substituted orunsubstituted pyridinyl ring).

In some examples, A is forosamine, B is 3,4,5-trimethoxyrhamnose, C isO, X¹ is N, X² is C(CH₃), and X³ is S. Optionally, the spinosyn compoundis(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13,10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione.

In some examples, A is forosamine, B is 3,4,5-trimethoxyrhamnose, C isO, X¹ is N, X² is C(NH₂), and X³ is S. Optionally, the spinosyn compoundis(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-amino-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione.

In some examples, A is forosamine, B is 3,4,5-trimethoxyrhamnose, C isO, R¹ is ethyl, X¹ is N, X² is (CCH₃), and X³ is S. Optionally, thespinosyn compound is(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.02,13.03,7.08,12]pentacosa-3(7),4,14-triene-16,24-dione.

In some examples, A is forosamine, B is 4-ethoxy-3,5-trimethoxyrhamnose,C is O, R¹ is ethyl, X¹ is N, X² is CR, X³ is S, and R is cyclopropyl.Optionally, the spinosyn compound is(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-cyclopropyl-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.02,13.03,7.08,12]pentacosa-3(7),4,14-triene-16,24-dione.

Also described herein are formulations. A formulation as describedherein comprises at least one spinosyn compound as described herein andan acceptable carrier. Optionally, the formulation can further compriseat least one additional active ingredient and/or at least one plant orplant product treatment compound. The at least one additional activeingredient can comprise, for example, an insecticide or a miticide(e.g., a contact-acting insecticide or contact-acting miticide).

Further described herein is a method for controlling pests. A method forcontrolling pests as described herein comprises contacting a pest withan effective amount of a spinosyn compound or a formulation as describedherein. Optionally, the pest is an insect, an arachnid, or a nematode.

Also described herein are methods for making a spinosyn compound. Amethod for making a spinosyn compound comprises reacting the C-5,6double bond of spinosyn A to form a spinosyn compound as describedherein, wherein the spinosyn compound forms via an α-halo ketoneintermediate.

The details of one or more embodiments are set forth in the drawings andthe description below. Other features, objects, and advantages will beapparent from the description and drawings, and from the claims.

DETAILED DESCRIPTION

Provided herein are spinosyn compounds. The compositions are useful inthe agricultural and animal health markets having activity towards pestssuch as insects, arachnids, nematodes and the like. Methods for makingthe compounds are also provided.

I. Compounds

A class of spinosyn compounds described herein is represented by FormulaI:

and salts thereof.

In Formula I,

is a single bond or a double bond.

Also, in Formula I, A is hydrogen or is selected from the groupconsisting of substituted or unsubstituted carbonyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. Optionally, A can be a substituted or unsubstitutedsaccharide. For example, A can be forosamine or a forosamine derivative,such as [(2R,5S,6R)-6-methyl-5-(methylamino)oxan-2-yl]oxy-.

Additionally, in Formula I, B is selected from the group consisting ofsubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. Optionally, B can be a substituted orunsubstituted saccharide. For example, B can be rhamnose or a rhamnosederivative, such as 3,4,5-trimethoxyrhamnose or4-ethoxy-3,5-dimethoxyrhamnose.

Further, in Formula I, C is O or NH.

Also, in Formula I, R¹ is substituted or unsubstituted alkyl orsubstituted or unsubstituted aryl. Optionally, R¹ is C₁-C₆ alkyl orC₁-C₆ aryl. Optionally, R¹ is ethyl.

Also, in Formula I, X¹, X², and X³ are each independently selected fromO, S, N, NR, CR, and CR₂, wherein each R is independently selected fromhydrogen, hydroxyl, substituted or unsubstituted amino, substituted orunsubstituted thio, substituted or unsubstituted alkoxy, substituted orunsubstituted aryloxy, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

In Formula I, when X¹ is O, X³ is not N.

Optionally, when X¹ and X² are selected from NR, CR, and CR₂, the Rgroups of X¹ and X² can be combined to form a substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. Optionally, when X² and X³ areselected from NR, CR, and CR₂, the R groups of X² and X³ can be combinedto form a substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkenyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

Optionally, the C14-C15 bond of the compound of Formula I is a doublebond. In these examples, Formula I can be represented by Structure I-A:

In Structure I-A, A, B, C, R¹, X¹, X², and X³ are as defined above forFormula I.

Optionally, X¹ is N, X² is CR, X³ is S, and the bonds (1) between X¹ andX², (2) between C3 and C7, and (3) between C14 and C15 of the compoundof Formula I are double bonds. In these examples, Formula I can berepresented by Structure I-B:

In Structure I-B, A, B, C, R¹, and R are as defined above for Formula I.

Optionally, X¹ is N, X² is CR, X³ is NH, and the bonds (1) between X¹and X², (2) between C3 and C7, and (3) between C14 and C15 of thecompound of Formula I are double bonds. In these examples, Formula I canbe represented by Structure I-C:

In Structure I-C, A, B, C, R¹, and R are as defined above for Formula I.

Optionally, X¹ is N, X² is CR, X³ is NR, and the bonds (1) between X¹and X², (2) between C3 and C7, and (3) between C14 and C15 of thecompound of Formula I are double bonds. In these examples, Formula I canbe represented by Structure I-D:

In Structure I-D, A, B, C, R¹, and R are as defined above for Formula I.

Optionally, X¹ is S, X² is CR, X³ is NH, and the bonds (1) between X¹and X², (2) between C3 and C7, and (3) between C14 and C15 of thecompound of Formula I are double bonds. In these examples, Formula I canbe represented by Structure I-E:

In Structure I-E, A, B, C, R¹, and R are as defined above for Formula I.

Optionally, A is hydrogen and the bonds (1) between X¹ and X², (2)between C3 and C7, and (3) between C14 and C15 of the compound ofFormula I are double bonds. In these examples, Formula I can berepresented by Structure I-F:

In Structure I-F, B, C, R¹, X¹, X², X³ and R are as defined above forFormula I.

Optionally, X¹ is N, X² is CR, X³ is NR, the R groups of X² and X³combine to form a cyclic structure, and the bonds (1) between X¹ and X²,(2) between C3 and C7, and (3) between C14 and C15 of the compound ofFormula I are double bonds. In these examples, Formula I can berepresented by Structure I-G:

In Structure I-G, A, B, C, R¹, and R are as defined above for Formula G.

Optionally, A is forosamine, B is 3,4,5-trimethoxyrhamnose, C is O, R¹is ethyl, X¹ is N, X² is C(CH₃), and X³ is S. The spinosyn compound canbe, for example,(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione.

Optionally, A is forosamine, B is 3,4,5-trimethoxyrhamnose, C is O, R¹is ethyl, X¹ is N, X² is C(NH₂), and X³ is S. The spinosyn compound canbe, for example,(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-amino-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione.

Optionally, A is forosamine, B is 4-ethyl-3,5-dimethoxyrhamnose, C is O,R¹ is ethyl, X¹ is N, X² is CR, R is alkyl, and X³ is S. The spinosyncompound can be, for example,(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.02,13.03,7.08,12]pentacosa-3(7),4,14-triene-16,24-dione.

Optionally, A is forosamine, B is 4-ethyl-3,5-dimethoxyrhamnose, C is O,R¹ is ethyl, X¹ is N, X² is CR, R is cycloalkyl, and X³ is S. Thespinosyn compound can be, for example,(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-cyclopropyl-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.02,13.03,7.08,12]pentacosa-3(7),4,14-triene-16,24-dione.

Examples of Formula I include the compounds shown in Table 1. In Table1, the compound number is shown to the left of the correspondingstructure.

TABLE 1 No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, the structures optionally alsoencompass the chemically identical substituents, which would result fromwriting the structure from right to left, e.g., —CH₂O— is intended toalso optionally recite —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di-, tri- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to optionally includethose derivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl.” Exemplary alkyl groups include the monounsaturatedC₉₋₁₀, oleoyl chain or the diunsaturated C_(9-10, 12-13) linoeyl chain.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The terms “aryloxy” and “heteroaryloxy” are used in their conventionalsense, and refer to those aryl or heteroaryl groups attached to theremainder of the molecule via an oxygen atom.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, S, Si and B,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. A heteroaryl group can beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to optionally include both substituted andunsubstituted forms of the indicated radical.

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocycloalkyl groups.

II. Methods of Making the Compounds

The compounds described herein can be prepared in a variety of ways. Thecompounds can be synthesized using various synthetic methods. At leastsome of these methods are known in the art of synthetic organicchemistry. The compounds described herein can be prepared from readilyavailable starting materials. Optimum reaction conditions can vary withthe particular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Variations on Formula I include the addition, subtraction, or movementof the various constituents as described for each compound. Similarly,when one or more chiral centers are present in a molecule, all possiblechiral variants are included. Additionally, compound synthesis caninvolve the protection and deprotection of various chemical groups. Theuse of protection and deprotection, and the selection of appropriateprotecting groups can be determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Wuts,Greene's Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons,2014, which is incorporated herein by reference in its entirety.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry(MS), or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

The compounds described herein can be prepared using spinosyn precursor,spinosyn, or spinosyn analogue starting materials, such as those setforth in U.S. Pat. No. 5,362,634. As used herein, spinosyn precursors,spinosyns, or spinosyn analogue starting materials used in the syntheticmethods include any tetracyclic spinosyn molecule comprising apolyketide-derived tetracyclic macrolide appended with two saccharides.

The methods of making the compounds described herein can include fromone to five chemical steps performed on spinosyns, often without needfor purification of thus formed intermediates.

Compounds described by Formula I and pharmaceutically acceptable saltsthereof can be made using the methods shown in Scheme 1, which depictsthe synthesis of compounds of Formula I wherein X¹ is N, X² is CR, andX³ is S.

In the synthetic method shown above in Scheme 1, spinosyn A can bebrominated using reagents such as bromine (Br₂) or N-bromosuccinimide(NBS) in the presence of water under basic or acidic conditions. Thebrominated intermediate can be oxidized using Dess-Martin periodinane orSwern conditions, followed by treatment of the intermediate with athioamide or thiourea.

Alternatively, compounds described herein can be made by those skilledin the art using synthetic chemistry transformations known to convert C,C double bonds to heterocyclic derivatives, either directly or inmultiple chemical steps. See, for example, Rakesh K. Parashar inChemistry of Heterocyclic Compounds, CRC Press, 2014, ISBN-13978-1466517 and John A. Joule and Keith Mills, Heterocyclic Chemistry,Wiley, 2010, ISBN-13 978-1405133005.

Additional modifications can be made to compounds according to Formula Iwhile retaining the desired activity of the compounds. For example, thesaccharide groups optionally present as A and B in the compoundsaccording to Formula I (e.g., forosamine and rhamnose) can be modifiedby methods in the art and retain pesticidal activity. Forosamine can bereplaced by certain nitrogen-containing sugars and non-sugarsubstituents with retention of some degree of activity. See, Gaisser etal. (2002) Chem. Comm. 6:618-619; and Gaisser et al. (2009) Org. Biomol.Chem. 7:1705-1708, herein incorporated by reference. Likewise, rhamnosereplacement analogs may be produced. See, Creemer et al. (2000) J.Antibiotics, 53:171-178; Sparks et al. (2001) Pest Manag. Sci.,57:896-905, herein incorporated by reference. Activity of the spinosynderivative can be retained after changes in the structure of therhamnose, especially certain modifications at C-2′ and C-3′ of thetri-O-methylrhamnose moiety.

Other methods of sugar modification can be made and are well known inthe art. See, Kirst et al. (2002) Curr. Top. Med. Chem. 2:675-699. Insome embodiments, one or more of the saccharide moieties is replacedwith another natural or a synthetic sugar. Synthetic sugars includemodified sugars. As used herein, a “modified sugar” is covalentlyfunctionalized with a “modifying group.” Useful modifying groupsinclude, but are not limited to, water-soluble polymers, therapeuticmoieties, diagnostic moieties, biomolecules and the like. Addition orremoval of any saccharide moieties present on the precursor or substrateis accomplished either chemically or enzymatically.

In some embodiments, chemical deglycosylation can be used by exposure ofthe spinosyn compounds described herein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theremainder of the molecule intact. See, Hakimuddin et al. (1987) Arch.Biochem. Biophys. 259:52 and Edge et al. (1981) Anal. Biochem. 118:131.Enzymatic cleavage of carbohydrate moieties on peptide variants can beachieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al. (1987) Meth. Enzymol. 138:350. Chemicaladdition of glycosyl moieties is carried out by any art-recognizedmethod. See, for example, U.S. Pat. Nos. 5,876,980; 6,030,815;5,728,554; 5,922,577; and WO 2004/99231.

Synthesis of the compounds of the invention is described below in theExamples. Generally, the methods comprise 1 to 5 chemical steps(semi-synthesis) performed on spinosyns or spinosyn precursors oftenwithout the need for purification of the intermediates. As indicatedherein, the terms “spinosyn precursors” or “spinosyns” used in themethod include any tetracyclic spinosyn molecule comprising apolyketide-derived tetracyclic macrolide appended with two saccharides.

III. Formulations

The compounds described herein or salts thereof can be provided in aformulation or composition. The spinosyn derivatives of the inventionmay be prepared in formulations or compositions for control of pests.The formulations can include other active ingredients and/or plant orplant product treatment compounds. Optionally, the formulation caninclude a contact-acting insecticide and/or miticide. Exemplarycontact-acting insecticides and/or miticides include those derived fromfatty acids, fatty acid esters, fatty acid sugar esters, and fatty acidsalts, pyrethrum extract, plant oils and their salts, vegetable oils andtheir salts, essential oils, mineral oils, pyrethrum extract, andcombinations thereof. The contact-acting insecticide and/or miticide canalso include avermectins. One skilled in the art will appreciate thatthe resulting spinosyn-containing compositions and formulationsdisclosed herein are not only pesticidally effective, but alsoenvironmentally sound and safe for human use. Further, some of thecompositions and formulations can be residual in that they do not leachout of baits or easily wash off of the leaves during rain, and thus canprotect against insect and mite pests during and after rainy weather.Optionally, the compositions and formulations can exhibit synergy, andresult in better than expected results than just the spinosyn or theinsecticide or miticide treatment alone.

Optionally, the present compounds are usefully combined withectoparasiticides (agents that control arthropod pests that typicallyattack their hosts on the external (“ecto”) surface). The spinosyncompounds are formulated for use as ectoparasiticides in manners knownto those skilled in the art. Representative ectoparasiticides includethe following: Abamectin, Alphamethrin, Amitraz, Avermectin, Coumaphos,Cycloprothrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyromazine,Deltamethrin, Diazinon, Diflubenzuron, Dioxathion, Doramectin, Famphur,Fenthion, Fenvalerate, Flucythrinate, Flumethrin, Hexaflumuron,Ivermectin, Lindane, Lufenuron, Malathion, Methoprene, Metriphonate,Moxidectin, Permethrin, Phosme, Pirimiphos, Propetamphos, Propoxur,Rotenone, Temephos, Tetrachlorvinphos, Trichlorfon, Zetacypermethrin,B.t. Biotoxins and Boric Acid.

Optionally, the present compounds are usefully combined with otherectoparasiticides or with anthelmentics, the latter also known asendoparasiticides (“endo”=internal, controlling internal parasites whichare typically platyhelminthes and nemathelminthes). Representative suchendoparasiticides include the following: Abamectin, Albendazole,Avermectin, Bunamidine, Coumaphos, Dichlorvos, Doramectin, Epsiprantel,Febantel, Fenbendazole, Flubendazole, Ivermectin, Levamisole,Mebendazole, Milbemycin, Morantel, Moxidectin, Netobimin, Niclosamide,Nitroscanate, Oxfendazole, Oxibendazole, Piperazine, Praziquantel,Pyrantel, Ricombendazole, Tetramisole, Thiabendazole, Clorsulon,Closantel, Diamphenethide, Nitroxynil, Oxyclozanide, Rafoxanide,Triclabendazole.

The formulations described herein can further include, in combinationwith the spinosyn component, one or more other compounds that haveactivity against the specific ectoparasite or endoparasite to becontrolled, such as, for example, synthetic pyrethroids, naturalpyrethins, organophosphates, organochlorines, carbamates, formamidines,avermectins, milbemycins, insect growth regulators (including chitinsynthesis inhibitors, juvenile hormone analogs, and juvenile hormones),nitromethylenes, pyridines and pyrazoles. In an exemplary embodiment,the composition can include an additional contact-acting insecticideand/or miticide. The compositions can be utilized as liquidconcentrates, Ready-To-Use (RTU) liquid sprays, dusts, or solids,depending upon the needs of the user. In use, the composition can beapplied to the pests themselves, in the vicinity of the pests, and/or inthe vicinity of plants and plant products that are to be protected.

In general, a formulation will include a compound as described hereinand one or more physiologically acceptable adjuvants. Formulationsinclude concentrated versions, in which the present active agent ispresent in a concentration of from 0.001 to 98.0 percent, with theremaining content being physiologically acceptable carriers. Suchformulations, especially those with less than 50 percent of the presentcompound, can sometimes be used directly, but these formulations canalso be diluted with other physiologically acceptable carriers to formmore dilute treating formulations. These latter formulations can includethe active agent in lesser concentrations of from 0.001 to 0.1 percent.

Compositions are prepared according to the procedures and formulas whichare conventional in the agricultural or pest control art. Thecompositions may be concentrated and dispersed in water or may be usedin the form of a dust, bait or granular formulation. The dispersions aretypically aqueous suspensions or emulsions prepared from concentratedformulations of the compounds. The water-soluble or water-suspension oremulsifiable formulations are either solids, wettable powders, orliquids, known as emulsifiable concentrates or aqueous suspensions.Wettable powders may be agglomerated or compacted to form waterdispersible granules. These granules comprise mixtures of compound,inert carriers and surfactants. The concentration of the compound istypically between about 0.1% to about 90% by weight. The inert carrieris typically attapulgite clays, montmorillonite clays and thediatomaceous earths or purified silicates.

Surfactants comprise typically about 0.5% to about 10% of the wettablepowder. Surfactants include sulfonated lignins, condensednapthalene-sulfonates, the napthalene-sulfonates,alkyl-benenesulfonates, alkysulfonates or nonionic surfactants such asethylene oxide adducts of alkylphenols or mixtures thereof. Emulsifiableconcentrates of the derivatives of the invention typically range fromabout 50 to about 500 grams of spinosyn derivative per liter of liquid,equivalent to about 10% to about 50%, dissolved in an inert carrierwhich is a mixture of a water immiscible solvent and emulsifiers.Organic solvents include organics such as xylenes, and petroleumfractions such as high-boiling naphthlenic and olefinic portions ofpetroleum which include heavy and aromatic naphtha. Other organics mayalso be used such as terpenic solvents—rosin derivatives, aliphaticketones such as cyclohexanone and complex alcohols. Emulsifiers foremulsifiable concentrates are typically mixed ionic and/or nonionicsurfactants such as those mentioned herein or their equivalents.

Aqueous suspensions may be prepared containing water-insoluble spinosynderivatives, where the compounds are dispersed in an aqueous vehicle ata concentration typically in the range of between about 5% to about 50%by weight. The suspensions are prepared by finely grinding the compoundand vigorously mixing it into a vehicle of water, surfactants, anddispersants. Inert ingredients such as inorganic salts and synthetic ornatural gums may also be employed to increase the density and/orviscosity of the aqueous vehicle as is desired.

Precipitated flowables may be prepared by dissolving at least onespinosyn derivative of the invention in a water-miscible solvent andsurfactants or surface active polymers. When these formulations aremixed with water, the active spinosyn derivative precipitates with thesurfactant controlling the size of the resulting micro-crystallineprecipitate. The size of the crystal can be controlled through theselection of specific polymer and surfactant mixtures.

The spinosyn derivatives may also be applied as a granular compositionthat is applied to the soil. The granular composition typically containsfrom about 0.5% to about 10% by weight of the derivative. The spinosynderivative is dispersed in an inert carrier which is typically clay oran equivalent substance. Generally, granular compositions are preparedby dissolving the compounds of the invention in a suitable solvent andapplying it to a granular carrier which has been pre-formed to thedesirable particle size. The particle size is typically between about0.5 mm to 3 mm. The granular compositions may also be prepared byforming a dough or paste of the carrier and compound, drying thecombined mixture, and crushing the dough or paste to the desiredparticle size.

The spinosyn derivative may also be combined with an appropriate organicsolvent. The organic solvent is typically a bland petroleum oil that iswidely used in the agricultural industry. These combinations aretypically used as a spray. More typically, the spinosyn compounds areapplied as a dispersion in a liquid carrier, where the liquid carrier iswater. The compounds may also be applied in the form of an aerosolcomposition. The compound is dissolved in an inert carrier, which is apressure-generating propellant mixture. The aerosol composition ispackaged in a container, where the mixture is dispersed through anatomizing valve. Propellant mixtures contain either low-boilinghalocarbons, which may be mixed with organic solvents or aqueoussuspensions pressurized with inert gases or gaseous hydrocarbons.

The compounds may be applied to any locus inhabited by an insect ormite. Such locus typically is cotton, soybean and vegetable crops, fruitand nut trees, grape vines, houses and ornamental plants. The amount ofthe spinosyn derivative applied to the loci of insects and mites can bedetermined by those skilled in the art. Generally, the concentrations offrom about 10 ppm to about 5,000 ppm provide the desired control. Forcrops such as soybeans and cotton, the rate of application is about 0.01to about 1 kg/ha, where the spinosyn derivative is applied in a 5 to 50gal/A spray formulation.

The composition can be formulated in a liquid concentrate, ready-to-use(RTU) liquid spray, dust, or solid form. The formulation chosen willdepend on the use of the product.

The following general treatment methods are preferably suitable forcarrying out the seed treatment, or plant propagation materialtreatment, according to the invention: dry treatments (preferably withaddition of adhesion promoters such as, for example, liquid paraffin ortalc), and, if appropriate, colorants, slurry treatments (preferablywith addition of wetters, dispersants, emulsifiers, adhesives, inertfillers and colorants), aqueous liquid treatments (preferably withaddition of emulsifiers, dispersants, thickeners, antifreeze agents,polymers, adhesives and colorants), solvent-based liquid treatments(with addition of solvents and colorants), emulsion treatments (withaddition of emulsifiers, solvents and colorants).

The total active spinosyn derivative in the treatment formulationspreferably amounts to 0.01% to 80% by weight. For example, the totalactive spinosyn compound can amount to 0.01% by weight, 0.05% by weight,0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% byweight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60%by weight, 70% by weight, or 80% by weight. Generally, about 1 to about300 g of spinosyn derivative are applied to every 100 kg of seed orplant propagation material in the form of a treatment.

Those of skill in the art will understand that the specific dose leveland frequency of dosage for any particular subject may be varied andwill depend upon a variety of factors, including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the species, age, body weight, general health, sex,and diet of the subject, the mode and time of administration, rate ofexcretion, drug combination, and severity of the particular condition.The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the infection, andshould be decided according to the judgment of the practitioner and eachsubject's circumstances.

IV. Methods of Use

The spinosyn compounds described herein have insecticidal and pesticidalactivity against pests, including insects, arachnids and nematodes.Therefore, the compounds and formulations as described herein can beused for controlling, inhibiting, and/or inactivating a pest ofinterest. The spinosyn compounds and formulations described hereinprovide a key source of agrichemicals with activities against crop pestspecies. In some instances, the compounds and formulations can be usedin animal health. The spinosyn compounds and formulations describedherein can be provided in agricultural and/or pharmaceuticalcompositions in effective amounts to control or inhibit the pest orcondition being treated. The spinosyn compounds and formulationsdescribed herein may possess one or more the following characteristicsas compared to natural spinosyns: increased potency; reduced risk tonon-target species; lower potential for environmental damage; minimalcross-resistance to other pesticides; and may overcome existing pestresistance to currently available spinosyn products.

The compounds and formulations described herein are useful incontrolling or containing pests populations. The compounds andformulations exhibit potent and broad-spectrum activity against numerouscommercially important insect pests. The spectrum of target insectsinclude many species of Lepidoptera and Diptera along with some membersof several other insect orders, including planthoppers, leafhoppers,spider mites and cockroaches. The compounds and formulations have potentand broad activity against many problematic larval species ofLepidoptera. Insecticidal activity is generally observed afteradministration of the spinosyns by a variety of delivery methods,including contact and oral feeding assays.

One skilled the art will appreciate that the compounds, formulations,and methods disclosed herein can be used to treat a variety of home andgarden insect and mite pests such as, by way of non-limiting example,members of the insect order Lepidoptera including Southern armyworm,codling moth, cutworms, clothes moths, Indian meal moth, leaf rollers,corn earworm, cotton bollworm (also called Tomato fruit worm), Europeancorn borer, imported cabbageworm, cabbage looper, pink bollworm,American bolloworm, tomato hornworm, bagworms, Eastern tent caterpillar,sod webworm, diamondback moth, tomato pinworm, grape berry moth, cottonleafworm, beet armyworm, and fall armyworm; members of the orderHomoptera including cotton aphid leafhoppers, plant hoppers, pearpsylla, scale insects, whiteflies, and spittle bugs; and members of theinsect order Diptera including house flies, stable flies, blow flies andmosquitoes; mites; and ants. The compounds and formulations describedherein can also be used to treat members of the order Thysanopteraincluding melon thrips and Western flower thrips; members of the orderColeoptera, including Colorado potato beetles; members of the orderOrthoptera; and Leaf miners of the orders Lepidoptera (moths andbutterflies), Hymenoptera (leaf mining sawflies), Coleoptera (beetles),and Diptera (true flies). The compounds and formulations can be used tocontrol and/or treat ants, green peach aphids, adult house flies,western tent caterpillar larvae, and two-spotted spider mites.Generally, the spinosyn compounds and formulations described herein canbe active against a number of ectoparasites in a number of animals by avariety of routes. The present compounds and formulations can be used tocontrol a wide variety of arthropod pests.

Representative pests which can be controlled by the present compoundsand formulations additionally include: Arachnids, Amblyomma americanum(Lone-star tick), Amblyomma maculatum (Gulf Coast tick), Argas persicus(fowl tick), Boophilus microplus (cattle tick), Chorioptes spp. (mangemite), Demodex bovis (cattle follicle mite), Demodex canis (dog folliclemite), Dermacentor andersoni (Rocky Mountain spotted fever tick),Dermacentor variabilis (American dog tick), Dermanyssus gallinae(chicken mite), Ixodes ricinus (common sheep tick), Knemidokoptesgallinae (deplumming mite), Knemidokoptes mutans (scaly-leg mite),Otobius megnini (ear tick), Psoroptes equi (scab mite), Psoroptes ovis(scab mite), Rhipicephalus sanguineus (brown dog tick), Sarcoptesscabiei (mange mite), Insects—Aedes (mosquitoes), Anopheles(mosquitoes), Culex (mosquitoes), Culiseta, Bovicola bovis (cattlebiting louse), Callitroga homnivorax (blowfly), Chrysops spp. (deerfly), Cimex lectularius (bed bug), Cochliomyia spp. (screwworm),Ctenocephalides canis (dog flea), Ctenocephalides felis (cat flea),Culicoides spp. (midges, sandflies, punkies, or no-see-ums), Damaliniaovis (sheep biting louse), Dermatobia spp. (warble fly), Gasterophilushaemorrhoidalis (nose bot fly), Gasterophilus intestinalis (common horsebot fly), Gasterophilus nasalis (chin fly), Glossina spp. (tsetse fly),Haematobia irritans (horn fly, buffalo fly), Haematopinus asini (horsesucking louse), Haematopinus eurysternus (short nosed cattle louse),Haematopinus ovillus (body louse), Haematopinus suis (hog louse),Hydrotaea irritans (head fly), Hypoderma bovis (bomb fly), Hypodermalineatum (heel fly), Linognathus ovillus (body louse), Linognathuspedalis (foot louse), Linognathus vituli (long nosed cattle louse),Lucilia spp. (maggot fly), Melophagus ovinus (sheep ked), Musca spp.(house fly, face fly), Oestrus ovis (nose bot fly), Pediculus spp.(lice), Phlebotomus spp. (sandfly), Phormia regina (blowfly), Psorophoraspp. (mosquito), Pthirus spp. (lice), Reduvius spp. (assassin bug),Simulium spp. (black fly), Solenopotes capillatus (little blue cattlelouse), Stomoxys calcitrans (stable fly), Tabanus spp. (horse fly),Tenebrio spp. (mealworms), Triatoma spp. (kissing bugs). Likewise, thespinosyn derivatives are useful against pests including: from the orderof the Isopoda, for example, Oniscus asellus, Armadillidium vulgare,Porcellio scaber; from the order of the Diplopoda, for example,Blaniulus guttulatus; from the order of the Chilopoda, for example,Geophilus carpophagus, Scutigera spp; from the order of the Symphyla,for example, Scutigerella immaculata.; from the order of the Thysanura,for example, Lepisma saccharina; from the order of the Collembola, forexample, Onychiurus armatus; from the order of the Orthoptera, forexample, Blatta orientalis, Periplaneta americana, Leucophaea maderae,Blattella germanica, Acheta domesticus, Gryllotalpa spp., Locustamigratoria migratorioides, Melanoplus differentialis, Schistocercagregaria; from the order of the Dermaptera, for example, Forficulaauricularia; from the order of the Isoptera, for example, Reticulitermesspp.; from the order of the Anoplura, for example, Phylloxera vastatrix,Pemphigus spp., Pediculus humanus corporis, Haematopinus spp.,Linognathus spp; from the order of the Mallophaga, for example,Trichodectes spp., Damalinea spp.; from the order of the Thysanoptera,for example, Frankliniella occidentalis, Hercinothrips femoralis, Thripspalmi, Thrips tabaci; from the order of the Heteroptera, for example,Eurygaster spp., Dysdercus intermedius, Piesma quadrata, Cimexlectularius, Rhodnius prolixus, Triatoma spp.; from the order of theHomoptera, for example, Aleurodes brassicae, Bemisia tabaci,Trialeurodes vaporariorum, Aphis gossypii, Brevicoryne brassicae,Cryptomyzus ribis, Aphis fabae, Doralis pomi, Eriosoma lanigerum,Hyalopterus arundinis, Macrosiphum avenae, Myzus spp., Phorodon humuli,Rhopalosiphum padi, Empoasca spp., Euscelis bilobatus, Nephotettixcincticeps, Lecanium corni, Saissetia oleae, Laodelphax striatellus,Nilaparvata lugens, Aonidiella aurantii, Aspidiotus hederae,Pseudococcus spp., Psylla spp.; from the order of the Lepidoptera, forexample, Pectinophora gossypiella, Bupalus piniarius, Cheimatobiabrumata, Lithocolletis blancardella, Hyponomeuta padella, Plutellamaculipennis, Malacosoma neustria, Euproctis chrysorrhoea, Lymantriaspp., Bucculatrix thurberiella, Phyllocnistis citrella, Agrotis spp.,Euxoa spp., Feltia spp., Earias insulana, Heliothis spp., Spodopteraexigua, Mamestra brassicae, Panolis flammea, Prodenia litura, Spodopteraspp., Trichoplusia ni, Carpocapsa pomonella, Pieris spp., Chilo spp.,Pyrausta nubilalis, Ephestia kuehniella, Galleria mellonella, Tineolabisselliella, Tinea pellionella, Hofmannophila pseudospretella, Cacoeciapodana, Capua reticulana, Choristoneura fumiferana, Clysia ambiguella,Homona magnanima, Tortrix viridana, Cnaphalocerus spp.; from the orderof the Coleoptera, for example, Anobium punctatum, Rhizopertha dominica,Acanthoscelides obtectus, Acanthoscelides obtectus, Hylotrupes bajulus,Agelastica alni, Leptinotarsa decemlineata, Phaedon cochleariae,Diabrotica spp., Psylliodes chrysocephala, Epilachna varivestis,Atomaria spp., Oryzaephilus surinamensis, Antho nomus spp., Sitophilusspp., Otiorrhynchus sulcatus, Cosmopolites sordidus, Ceuthorrhynchusassimilis, Hypera postica, Dermestes spp., Trogoderma spp., Anthrenusspp., Attagenus spp., Lyctus spp., Meligethes aeneus, Ptinus spp.,Niptus hololeucus, Gibbium psylloides, Tribolium spp., Tenebrio molitor,Agriotes spp., Conoderus spp., Melolontha melolontha, Amphimallonsolstitialis, Costelytra zealandica, Oulema oryzae, Lissorhoptrusoryzophilus; from the order of the Hymenoptera, for example, Diprionspp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Vespa spp.;from the order of the Diptera, for example, Aedes spp., Anopheles spp.,Culex spp., Drosophila melanogaster, Musca spp., Fannia spp., Calliphoraerythrocephala, Lucilia spp., Chrysomyia spp., Cuterebra spp.,Gastrophilus spp., Hyppobosca spp., Liriomyza spp., Stomoxys spp.,Oestrus spp., Hypoderma spp., Tabanus spp., Tannia spp., Bibiohortulanus, Oscinella frit, Phorbia spp., Pegomyia hyoscyami, Ceratitiscapitata, Dacus oleae, Tipula paludosa; from the order of theSiphonaptera, for example, Xenopsylla cheopis, Ceratophyllus spp.; fromthe order of the Arachnida, for example, Scorpio maurus, Latrodectusmactans; from the order of the Acarina, for example, Acarus siro, Argasspp., Ornithodoros spp., Dermanyssus gallinae, Eriophyes ribis,Phyllocoptruta oleivora, Boophilus spp., Rhipicephalus spp., Amblyommaspp., Hyalomma spp., Ixodes spp., Psoroptes spp., Chorioptes spp.,Sarcoptes spp., Tarsonemus spp., Bryobia praetiosa, Panonychus spp.,Tetranychus spp.

Insects that can be controlled with the aid of the compounds andformulations described herein include those of the following orders:soil-dwelling insects: Diptera (for example the frit-fly, wheat-bulbfly), Coleoptera (for example Diabrotica (wire worm), Lepidoptera (forexample dart moth), Blattophtheroidea, Myriopoda. Leaf insects:Aphidina, Coleoptera, Brachycera, Lepidotera, Homoptera, Tysanoptera,Aleurodina, Cicadina, Acasi, Cossina, Heteroptera.

Methods for controlling insect and mite pests as described herein caninclude providing a formulation that has an effective amount of at leastone spinosyn compound as described herein, at least one of an additionalinsecticide and miticide, and at least one of a solvent or an acceptablecarrier, and administering an effective amount of the formulation tocontrol pests. Where the formulation is a liquid, the method can furtherinclude administering an effective amount of the formulation such thatan effective amount of the formulation contacts pests, plants and plantproducts, the vicinity of the pests, and/or the vicinity of the plantsand plant products. Where the formulation is a dust or a solid,administering an effective amount of the formulation can include placingan effective amount of the composition in a vicinity of pests and/orplacing an effective amount of the composition in a vicinity of plantsand plant products to be protected.

An effective amount of the spinosyn compound or formulation as describedherein is an amount to control or kill the target pest. The use ratesvary widely and are highly impacted by the target pest, target pest sizeand number, host crop and crop age, climate and economic threshold oracceptable damage. In general, a typical use rate is set at about 1 ppm(1 mg a.i./kg of grain). For use on crops, between about 25 and about200 grams per hectare (0.023 and 0.184 lbs per acre) of activeingredient is used. Turf rates are 88-450 g a.i./ha (0.078-0.4 lbai/acre). Ornamental rates are 0.046-0.17 lb ai/100 gallons or 55-204ppm. There is typically a positive temperature correlation that resultsin better activity with higher temperatures. Performance against somepests, such as leafminers and thrips, are positively impacted by theaddition of nominal rates of penetrating surfactants such as crop oils.

All animals are subject to attack by such pests, though the problems aremost severe among vertebrate hosts. Accordingly, the spinosyn compoundsand formulations described herein can be used on humans, livestockanimals, (cattle, sheep, pigs, goats, buffalo, water buffalo, deer,rabbits, chickens, turkeys, ducks, geese, ostriches, and the like),horses and other pleasure animals, mink and other animals grown fortheir fur, rats, mice, other animals used in laboratory and researchsettings, companion animals such as dogs and cats, fish, crustacea, andother aquatic animals. In short, the spinosyn compounds and formulationsdescribed herein are useful for treatment of the whole range of animals.

Arthropod pests are inhibited or killed on a host animal by contactingthe pest with an effective amount of a spinosyn compound as describedherein.

Techniques for delivering the compounds and formulations describedherein are well known to those skilled in the art. In general, a presentformulation comprising at least one spinosyn compound is applied to theexterior surface of an animal, whereby it contacts pests already presenton the host as well as those which arrive on the host's body within theefficacy period. Typically, the spinosyn compound is formulated in aliquid formulation which is sprayed onto the animal's surface or pouredonto the animal's surface. Another conventional treatment is a “dip”,whereby cattle are treated by being substantially immersed in a dilutesolution containing the spinosyn compound. For some hosts and pests, theformulation can be a dust, which is sprinkled onto the host, or ashampoo or cream which is employed in bathing the animal. Collars oncats and dogs can also be employed as a way of delivering thederivatives directly to the animal's surface.

The compounds and formulations described herein can also be applied tolocations frequented by animals, so that pests are thereby contacted bythe compound even as in direct application to the host. Application topet bedding can be used, as well as application to carpeting. Forcattle, dusting bags can be used. These are positioned in a doorwaywhere the cattle inevitably rub against the bag and pests are contactedby the present compound.

Optionally, the present compounds and formulations can be used tocontrol insects and arachnids which are pests in the feces of cattle andother animals. The compounds and formulations can be administered orallyand the compounds travel through the intestinal tract and emerge in thefeces. Control of pests in the feces indirectly protects the animalsfrom the pests.

The compounds and formulations described herein may be applied to thefoliage of a plant which a pest might feed on. Additionally, thecompounds may be used orally or topically to control pests on animals.

Oral administration may be carried out using tablets and animal feeds.For some animals, such as certain cats, administration is bestaccomplished by using an acceptable liquid formulation that isadministered directly or added to their food ration. Especially usefulmethods of orally administering the spinosyn derivatives are byadministering it in chewable tablets or treats and animal feeds.

The spinosyn compounds and formulations described herein are also usefulfor the treatment of animals to control arthropods, i.e., insects andarachnids, which are pests on animals. These arthropod pests typicallyattack their hosts on the external (“ecto”) surface; agents whichcontrol such pests are referred to as “ectoparasiticides”.

The spinosyn compounds and formulations can be used for treating thesoil, for treating seed or plant propagation material, and for drenchingand irrigating plants. The following exemplary types of seed and plantpropagation material can be treated: maize, cereals (such as, forexample, wheat, barley, oats, rye), rice, seed potatoes, cotton, oilseedrape, sunflower, beet (such as, for example, sugar beet), vegetable seed(such as, for example, onion, cabbage, tomato), (fodder) legumes,peanuts, soya, sorghum, and the like.

It is advantageous to apply granules comprising the active compounddescribed herein into or onto the soil. Examples of suitableapplications include broadcast, band, furrow and planting-holeapplication.

It is particularly advantageous to emulsify or dissolve the spinosyns ortheir salts in water and to use this for irrigating the plants. Examplesof suitable applications are spraying onto the soil, drenching, i.e.irrigating the plants with active-compound-containing solutions, anddrip irrigation, and also use in hydroponic systems, in particular inthe production of vegetables and ornamentals.

Seed treatments are suitable for controlling animal pests, preferablyarthropods and nematodes, in particular insects and arachnids, which arefound in agriculture and in forests. They are effective againstnormally-sensitive and resistant species and against all or individualdevelopmental stages.

In some embodiments, the spinosyn compounds and formulations describedherein can be used for promoting or accelerating wound healing in amammal comprising administering at least one spinosyn compound or aphysiologically acceptable derivative or salt thereof, to a mammal inneed thereof. In this manner, the spinosyn compounds and formulationscan be used for the manufacture of a medicament for promoting oraccelerating wound healing in animals, including humans (see, forexample, U.S. Pat. No. 8,536,142) or in the treatment of head lice inhumans.

As used herein the terms treatment, treat, or treating refer to a methodof reducing one or more symptoms of a disease, infection, or condition.Such methods include controlling, inhibiting, and/or inactivating apest. Thus in the disclosed method, treatment can refer to a 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in pests found incrops or animals and/or the severity of one or more symptoms of thedisease, infection, or condition associated with such pests. Forexample, a method for controlling a pest is considered to be a treatmentif there is a 10% reduction in one or more pests in a crop or in asubject as compared to a control. Similarly, a method for treating aninfection is considered to be a treatment if there is a 10% reduction inone or more symptoms or signs of an infection in a subject as comparedto a control. As used herein, control refers to the untreated condition.Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or any percent reduction in between 10% and 100% as compared tonative or control levels. It is understood that treatment does notnecessarily refer to a complete elimination of pests, or a cure orcomplete ablation of the disease, infection, condition, or symptoms ofthe disease, infection, or condition.

As used herein, subject means both mammals and non-mammals. Mammalsinclude, for example, humans; non-human primates, e.g., apes andmonkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammalsinclude, for example, fish and birds.

Non-limiting embodiments include:

1. A spinosyn compound of the following formula:

or a salt thereof, wherein:

is a single bond or a double bond;

A is hydrogen or is selected from the group consisting of substituted orunsubstituted carbonyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl;

B is selected from the group consisting of substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl;

C is O or NH;

R¹ is C₁-C₆ alkyl or C₁-C₆ aryl;

X¹, X², and X³ are each independently selected from O, S, N, NR, CR, andCR₂, wherein each R is independently selected from hydrogen, hydroxyl,substituted or unsubstituted amino, substituted or unsubstituted thio,substituted or unsubstituted alkoxy, substituted or unsubstitutedaryloxy, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl,

wherein when X¹ is O, X³ is not N.

2. The embodiment of paragraph 1, wherein when X¹ and X² are selectedfrom NR, CR, and CR₂, the R groups of X¹ and X² combine to form asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl,and substituted or unsubstituted heteroaryl.

3. The embodiment of paragraph 1, wherein when X² and X³ are selectedfrom NR, CR, and CR₂, the R groups of X² and X³ combine to form asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl,and substituted or unsubstituted heteroaryl.

4. The embodiment of any of paragraphs 1-3, wherein A comprisesforosamine or a forosamine derivative.

5. The embodiment of any of paragraphs 1-4, wherein B comprises a[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy or a(2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy group.

6. The embodiment of any of paragraphs 1-5, wherein A is forosamine, Bis a [(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy or a(2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy group, C is O, R¹is ethyl, X¹ is N, X² is CR, R is alkyl, and X³ is S.

7. The embodiment of any of paragraphs 1-6, wherein the spinosyncompound is(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione.

8. The embodiment of any of paragraphs 1-5, wherein A is forosamine, Bis a [(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy or a(2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy group, C is O, R¹is ethyl, X¹ is N, X² is CR, R is amino, and X³ is S.

9. The embodiment of any of paragraphs 1-5 or 8, wherein the spinosyncompound is(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-amino-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione.

10. The embodiment of any of paragraphs 1-5, wherein A is forosamine, Bis a (2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy group, C is O,R¹ is ethyl, X¹ is N, X² is CR, R is alkyl, and X³ is S.

11. The embodiment of any of paragraphs 1-5 or 10, wherein the spinosyncompound is(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.02,13.03,7.08,12]pentacosa-3(7),4,14-triene-16,24-dione.

12. The embodiment of any of paragraphs 1-5, wherein A is forosamine, Bis a (2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy group, C is O,R¹ is ethyl, X¹ is N, X² is CR, R is cyclopropyl, and X³ is S.

13. The embodiment of any of paragraphs 1-5 or 12, wherein the spinosyncompound is(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-cyclopropyl-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.02,13.03,7.08,12]pentacosa-3(7),4,14-triene-16,24-dione

14. The embodiment of any of paragraphs 1 or 3-13 wherein the C14-C15bond of the compound of Formula I is a double bond.

15. The embodiment of any of paragraphs 1, 3-13 or 14 having the formularepresented by Structure I-A:

16. The embodiment of any of paragraphs 1 or 3-13, wherein X¹ is N, X²is CR, X³ is S, and the bonds (1) between X¹ and X², (2) between C3 andC7, and (3) between C14 and C15 of the compound of Formula I are doublebonds.

17. The embodiment of any of paragraphs 1, 3-13 or 16, having theformula represented by Structure I-B:

18. The embodiment of any of paragraphs 1 or 3-5, wherein X¹ is N, X² isCR, X³ is NH, and the bonds (1) between X¹ and X², (2) between C3 andC7, and (3) between C14 and C15 of the compound of Formula I are doublebonds.

19. The embodiment of any of paragraphs 1, 3-5 or 18, having the formularepresented by Structure I-C:

20. The embodiment of any of paragraphs 1 or 3-5, wherein X¹ is N, X² isCR, X³ is NR, and the bonds (1) between X¹ and X², (2) between C3 andC7, and (3) between C14 and C15 of the compound of Formula I are doublebonds.

21. The embodiment of any of paragraphs 1, 3-5 or 20, having the formularepresented by Structure I-D:

22. The embodiment of any of paragraphs 1 or 3-5, wherein X¹ is S, X² isCR, X³ is NH, and the bonds (1) between X¹ and X², (2) between C3 andC7, and (3) between C14 and C15 of the compound of Formula I are doublebonds.

23. The embodiment of any of paragraphs 1, 3-5 or 22, having the formularepresented by Structure I-E:

24. The embodiment of any of paragraphs 1, 3 or 5, wherein A is hydrogenand the bonds (1) between X¹ and X², (2) between C3 and C7, and (3)between C14 and C15 of the compound of Formula I are double bonds.

25. The embodiment of any of paragraphs 1, 3, 5 or 24, having theformula represented by Structure I-F:

26. The embodiment of any of paragraphs 1 or 3-5, wherein X¹ is N, X² isCR, X³ is NR, the R groups of X² and X³ combine to form a cyclicstructure, and the bonds (1) between X¹ and X², (2) between C3 and C7,and (3) between C14 and C15 of the compound of Formula I are doublebonds.

27. The embodiment of any of paragraphs 1, 3-5 or 26, having the formularepresented by Structure I-G:

28. A formulation, comprising at least one spinosyn compound of any ofparagraphs 1-27 and an acceptable carrier.

29. The embodiment of paragraph 28, further comprising at least oneadditional active ingredient.

30. The embodiment of paragraph 28 or 29, further comprising at leastone plant or plant product treatment compound.

31. The embodiment of paragraph 29, wherein the at least one additionalactive ingredient comprises an insecticide or a miticide.

32. The embodiment of paragraph 31, wherein the insecticide is acontact-acting insecticide.

33. The embodiment of paragraph 31, wherein the miticide is acontact-acting miticide.

34. A method for controlling pests, comprising contacting a pest with aneffective amount of a spinosyn compound of any of paragraphs 1-27 or aformulation of any of paragraphs 28-33.

35. The embodiment of paragraph 34, wherein the pest is an insect.

36. The embodiment of paragraph 34, wherein the pest is an arachnid.

37. The embodiment of paragraph 34, wherein the pest is a nematode.

38. A method for making a spinosyn compound, comprising reacting theC-5,6 double bond of Spinosyn A to form a spinosyn compound according toparagraph 1, wherein the spinosyn compound forms via an α-halo ketoneintermediate.

The routes below illustrate general methods of synthesizing compounds ofFormula (I) and/or pharmaceutically acceptable salts thereof. Theskilled artisan will appreciate that the compounds described hereincould be made by methods other than those specifically described herein,by adaptation of the methods described herein and/or by adaptation ofmethods known in the art. For example, compounds described herein can bemade using other spinosyn derivatives (e.g. spinetoram, spinosyn J,spinosyn L, or others) as starting materials. In general, compoundsprovided herein may be prepared in a multi-step synthesis, as shownbelow. All quantities shown are approximate, and are given solely forillustrative purposes.

The examples below are intended to further illustrate certain aspects ofthe methods and compounds described herein, and are not intended tolimit the scope of the claims.

EXAMPLES Intermediate 1:(2S,3aR,5aR,5bS,9S,13S,14R,16aR,16bS)-4-bromo-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-9-ethyl-14-methyl-2-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-1H,2H,3H,3aH,4H,5H,5aH,5bH,6H,7H,9H,10H,-11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-5,7,15-trione

To a solution of Spinosyn A (5.0 g, 6.8 mmol) in dimethyl sulfoxide(DMSO, 50 mL) was added dropwise 10 mL of water and concentratedsulfuric acid (670 mg, 6.8 mmol). The mixture was then cooled to 0° C.and N-bromosuccinimide (NBS; 1.2 g, 6.8 mmol) was added. After stirringfor 30 minutes at 0° C., ethyl acetate (200 mL) and saturated aqueoussodium bicarbonate (150 mL) were added. The organic layer was washedwith water and brine, dried over sodium sulfate, filtered andconcentrated in vacuo to give Intermediate 5, a white solid (5.5 g,97.1%), which was used in the next chemical step without furtherpurification.

To a solution of this white solid (5.5 g, 6.6 mmol) in dichloromethane(100 mL) at 0° C. was added Dess-Martin periodinane (DMP) (3.1 g, 7.3mmol). The mixture was stirred at room temperature overnight. Themixture was then washed with saturated sodium bicarbonate (30 mL),saturated Na₂SO₃ (20 mL), and brine. The organic layer was dried oversodium sulfate, filtered, and concentrated in vacuo to give an oil whichwas purified by silica gel column chromatography (using adichloromethane (dichloromethane) to methanol (methanol) gradient offrom 50/1 to 15/1) to afford the title compound (2.1 g, 38.6%) as awhite solid.

Intermediate 2:(2S,3aR,5aR,5bS,9S,13S,14R,16aR,16bS)-5-bromo-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-9-ethyl-14-methyl-2-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-1H,2H,3H,3aH,4H,5H,5aH,5bH,6H,7H,9H,10H,11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-4,7,15-trione

To a solution of Intermediate 5 (600 mg, 0.72 mmol) in DCM (20 mL) wasadded DMP (368 mg, 0.87 mmol) at 0° C. The mixture was stirred at r.t.for 2 h. Water (30 mL) was added, and the mixture was extracted withdichloromethane (20 mL×2). The organic layer was separated, dried oversodium sulfate, filtered and concentrated to dryness. The residue waspurified by silica gel chromatography (dichloromethane:methanol100:1˜20:1) to afford Intermediate 2 (150 mg, yield 25%) as a whitesolid.

Example 1:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-amino-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]-pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (400 mg, 0.48 mmol) in dimethylformamide(DMF; 3 mL) was added thiourea (73 mg, 0.96 mmol). The mixture was thenheated to 80° C. under microwave for 12 minutes. After cooling to roomtemperature, the mixture was purified by flash column chromatography toafford the title compound (162 mg, 27.5%) as a white solid. Partial ¹HNMR (DMSO-d₆, 400 MHz): δ7.10 (s, 1H), 6.73 (brs, 2H), 4.81 (s, 1H),4.61-4.61 (m, 1H), 4.45-4.44 (m, 1H), 4.32-4.30 (m, 1H), 3.80 (t, J=8.4Hz, 1H), 3.02-2.92 (m, 5H), 0.74 (t, J=6.8 Hz, 1H). LCMS: m/z 803.9[M+H]⁺.

Example 2:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (400 mg, 0.48 mmol) in DMF (3 mL) wasadded thioacetamide (72 mg, 0.96 mmol). The mixture was then heated to120° C. under microwave for 1 hour. After cooling to room temperature,the mixture was purified by chromatography over silica gel to afford thetitle compound (46 mg, 11.8%) as a white solid. Partial ¹H NMR (DMSO-d₆,400 MHz): δ7.15 (s, 1H), 4.83 (s, 1H), 4.63-4.59 (m, 1H), 4.44-4.40 (m,1H), 4.34-4.33 (m, 1H), 4.05-4.01 (m, 1H), 3.53 (s, 1H), 3.10-2.93 (m,5H), 2.79-2.72 (m, 1H), 2.60 (s, 3H), 0.76 (t, J=7.2 Hz, 3H). LCMS: m/z802.9 [M+H]⁺.

Example 3:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-5,22-diethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (500 mg, 0.60 mmol) and propionamidinehydrochloride (131 mg, 1.21 mmol) in acetonitrile (10 mL) was addedpotassium carbonate (250 mg, 1.81 mmol). The mixture was then heated to85° C. for 6 hours. The mixture was evaporated under reduced pressure togive an oil, which was purified by silica gel chromatography using adichloromethane/methanol gradient of from 50/1 to 15/1 to give the crudeproduct. The crude product was further purified by preparative HPLC toafford the title compound (53 mg, yield 10.9%) as a white solid. Partial¹H NMR (DMSO-d₆, 400 MHz): δ7.14 (s, 1H), 4.83 (s, 1H), 4.65-4.61 (m,1H), 4.44 (d, J=9.2 Hz, 1H), 3.92 (t, J=8.4 Hz, 1H), 2.98-2.93 (m, 5H),2.68 (q, J=7.2 Hz, 2H), 0.77 (t, J=7.2 Hz, 1H). LCMS: m/z 799.9 [M+H]⁺.

Example 4:(1R,15R,17S,19S,20R,24R,25S,29S,33S)-25-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-29-ethyl-24-methyl-17-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-30-oxa-3,13-diazaheptacyclo[18.13.0.0^(2,14).0^(4,13).0^(5,10).0^(15,19).0^(22,33)]tritriaconta-2(14),3,5(10),6,8,11,21-heptaene-23,31-dione

A mixture of Intermediate 1 (250 mg, 0.3 mmol) and quinolin-2-ylamine(87 mg, 0.6 mmol) in t-BuOH (2 mL) was heated by microwave at 85° C. for3 h. The mixture was purified by prep-HPLC to afford the title compound(15 mg, yield 5.7%) as a yellow solid. Partial ¹H NMR (CDCl₃, 400 MHz):δ8.63 (d, J=8.0 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H), 7.66 (d, J=8.0 Hz, 1H),7.59 (t, J=8.0 Hz, 1H), 7.52 (dt, J=6.0, 1.2 Hz, 1H), 6.99 (d, J=6.8 Hz,1H), 6.85 (s, 1H), 4.96 (s, 1H), 4.86-4.77 (m, 1H), 4.54-4.48 (m, 1H),4.45-4.36 (m, 2H), 3.44-3.38 (m, 2H), 3.36-3.26 (m, 2H), 3.21-3.12 (m,2H), 3.10-3.01 (m, 1H), 2.56-2.49 (m, 1H), 2.41-2.32 (m, 1H), 2.25 (s,7H), 0.88 (t, J=7.6 Hz, 3H). LCMS: m/z 871.9 [M+H]⁺.

Example 5:(1S,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(phenylamino)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded phenylthiourea (170 mg, 1.1 mmol) and the resulting mixture wasstirred at reflux overnight. The reaction mixture was concentrated andthe residue was purified by prep-HPLC to afford the title compound (150mg, 17.0% yield) as a white solid. Partial ¹H NMR (400 MHz, Acetone-d₆):δ 7.54 (d, J=8.4 Hz, 2H), 7.17 (t, J=8.0 Hz, 2H), 6.99 (s, 1H), 6.81 (t,J=7.2 Hz, 2H), 4.75 (d, J=1.6 Hz, 1H), 4.63-4.58 (m, 1H), 4.36-4.30 (m,2H), 4.04 (m, 1H), 3.07-3.05 (m, 2H), 3.01-2.96 (m, 1H), 2.90 (t, J=9.2Hz, 1H), 2.69-2.62 (m, 2H), 2.36-2.27 (m, 1H), 2.08 (s, 6H), 2.06-1.97(m, 2H), 1.09-1.06 (m, 7H), 1.01 (d, J=6.8 Hz, 3H), 0.72 (t, J=7.2 Hz,3H); LCMS: m/z 881.1 [M+H]⁺.

Example 6:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-5,22-diethyl-6,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a stirred solution of Example 3 in DMF (5 mL) was added sodiumhydride (60% in mineral oil, 38 mg, 0.93 mmol) at 0° C. After stirringfor 5 min, iodomethane (89 mg, 0.62 mmol) was added. The mixture wasstirred at room temperature for 2 h. The mixture was diluted with ethylacetate (50 mL), washed with saturated ammonium chloride aqueous (30mL), water (20 mL), and brine. The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (39 mg, 7.6% yield)as a white solid. Partial ¹H NMR (CDCl₃, 400 MHz): δ6.74 (s, 1H), 4.85(d, J=1.2 Hz, 1H), 4.73-4.69 (m, 1H), 4.43-4.38 (m, 2H), 4.12-4.08 (m,1H), 3.27-3.22 (m, 3H), 3.13-3.09 (m, 2H), 3.00-2.96 (m, 1H), 2.85-2.83(m, 2H), 2.72-2.64 (m, 1H), 2.39 (s, 6H), 2.29-2.24 (m, 2H), 0.80 (t,J=7.6 Hz, 3H). LCMS: m/z 813.9 [M+H]⁺.

Example 7:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-phenyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (500 mg, 0.48 mmol) in ethanol (10 mL)was added thiobenzamide (124 mg, 0.90 mmol). The mixture was refluxedovernight, then heated at 120° C. under microwave for 10 min. Themixture was concentrated and purified by Prep-HPLC to afford the titlecompound (130 mg, 24.8%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ7.91-7.89 (m, 2H), 7.50-7.43 (m, 3H), 7.17 (s, 1H), 4.86 (s, 1H),4.68-4.62 (m, 1H), 4.43-4.34 (m, 2H), 4.16 (t, J=8.8 Hz, 1H), 3.59-3.31(m, 17H), 3.20-3.05 (m, 4H), 2.97 (t, J=9.2 Hz, 1H), 2.91-2.84 (m, 1H),2.40-2.34 (m, 1H), 2.26-2.21 (m, 1H), 2.15 (s, 6H), 2.14-1.13 (m, 20H),1.08 (d, J=6.4 Hz, 3H), 0.79 (t, J=7.2 Hz, 3H). LCMS: m/z 865.8 [M+H]⁺.

Example 8:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-phenyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (1.0 g, 1.21 mmol) and benzamidinehydrochloride (379 mg, 2.42 mmol) in acetonitrile (10 mL) was addedpotassium carbonate (501 mg, 3.63 mmol). The mixture was then heated to85° C. for 2 h. The mixture was concentrated under reduced pressure togive an oil which was purified by chromatography on silica gel column(dichloromethane/methanol=50/1 to 15/1) to give the crude product. Thecrude product was further purified by prep-HPLC to afford the titlecompound (260 mg, yield 25%) as a white solid. Partial ¹H NMR (DMSO-d₆,400 MHz): δ12.15-11.99 (m, 1H), 7.96-7.89 (m, 2H), 7.42-7.39 (m, 2H),7.30-7.28 (m, 1H), 7.19-7.14 (m, 1H), 4.86 (d, J=13.2 Hz, 1H), 4.71-4.65(m, 1H), 4.44-4.35 (m, 2H), 4.11-3.89 (m, 1H), 3.55-3.29 (m, 29H),3.05-2.93 (m, 5H), 2.72-2.68 (m, 1H), 2.33-1.07 (m, 24H), 0.80-0.77 (m,1H). LCMS: m/z 848.4 [M+H]⁺.

Example 9:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-6,22-diethyl-17-methyl-5-phenyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione

To a stirred solution of Example 8 (1.0 g, 1.18 mmol) in DMF (15 mL) wasadded sodium hydride (60% in mineral oil, 34 mg, 1.42 mmol) underice-water bath. After stirring for 10 min, iodoethane (276 mg, 1.77mmol) was added. The mixture was stirred at room temperature for 1 h,and then diluted with ethyl acetate (50 mL), washed with saturatedammonium chloride (30 mL), water (20 mL), and brine. The organic layerwas dried over sodium sulfate, filtered and concentrated. The residuewas purified by prep-HPLC to afford the title compound (50 mg, 4.8%yield) as a white solid. Partial ¹H NMR ¹H NMR (CDCl3, 400 MHz):7.56-7.54 (m, 2H), 7.45-7.36 (m, 3H), 6.81 (s, 1H), 4.90 (s, 1H),4.78-4.72 (m, 1H), 4.47-4.41 (m, 2H), 4.17-4.03 (m, 2H), 3.95-3.88 (m,1H), 3.70-3.45 (m, 15H), 3.33-3.26 (m, 2H), 3.20-2.12 (m, 3H), 3.05-2.99(m, 1H), 2.86-2.78 (m, 1H), 2.34-2.18 (m, 9H), 2.01-1.69 (m, 8H), 0.82(t, J=7.6 Hz, 3H). LCMS: m/z 876.4 [M+H]⁺.

Example 10:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(4-bromophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added 4-Bromo-thiobenzamide (470 mg, 2.1 mmol). The mixture wasrefluxed overnight and then heated at 120° C. under microwave for 1 h.After concentration, the residue was dissolved in ethyl acetate (100 mL)and washed with saturated sodium bicarbonate aqueous (50 mL). Thecombined organic layer was dried over anhydrous sodium sulfate, filteredand concentrated. The residue was purified by chromatography on silicagel column (dichloromethane/methanol=30/1 to 15/1) to afford the crudeproduct which was further purified by prep-HPLC to afford the titlecompound (130 mg, 12.6% yield) as a white solid. Partial ¹H NMR (CDCl₃,300 MHz): δ7.77 (d, J=8.4 Hz, 2H), 7.52 (d, J=8.7 Hz, 2H), 6.79 (s, 1H),4.89 (d, J=0.6 Hz, 1H), 4.78-4.73 (m, 1H), 4.48-4.32 (m, 3H), 2.93-2.84(m, 1H), 2.40-2.34 (m, 1H), 0.85 (t, J=7.5 Hz, 3H). LCMS: m/z 943.9,945.9 [M+H]⁺.

Example 11:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-(4-hydroxyphenyl)-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added 4-Hydroxy-thiobenzamide (333 mg, 2.1 mmol). The mixture wasrefluxed overnight and allowed to cool to room temperature. The mixturewas concentrated and purified by flash column to afford the titlecompound (370 mg, 38.6%) as a white solid. Partial ¹H NMR (CDCl₃, 300MHz): δ7.76 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.7 Hz, 2H), 6.80 (d, J=0.9Hz, 1H), 4.89 (s, 1H), 4.78-4.72 (m, 1H), 4.47-4.41 (m, 2H), 4.29 (t,J=8.1 Hz, 1H), 3.66-3.08 (m, 20H), 2.91-2.81 (m, 1H), 2.41-2.17 (m, 9H),0.84 (t, J=7.2 Hz, 3H). LCMS: m/z 881.1 [M+H]⁺.

Examples 12 and 32:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-(4-hydroxyphenyl)-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(12), and(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(propylsulfanyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(13)

To a solution of intermediate 1 (1.6 g, 1.9 mmol) in ethanol (12 mL) wasadded ammonium dithiocarbamate (418 mg, 3.8 mmol). The mixture washeated to 75° C. for 1 h with an oil bath, then heated to 100° C. undermicrowave for 30 min. The mixture was concentrated in vacuo to giveExample 12 (2.0 g, crude) as a yellow solid, which was used in next stepwithout further purification.

To a solution of Example 12 (500 mg, crude, 0.60 mmol) and n-PrBr (225mg, 1.82 mmol) in acetonitrile (10 mL) was added potassium carbonate(420 mg, 3.0 mmol). The mixture was heated to 80° C. for 2 h. Themixture was evaporated under reduced pressure, and the residue wasdiluted with ethyl acetate (60 mL), washed with water (50 mL) and thenbrine, dried over sodium sulfate, filtered and concentrated. The residuewas purified by prep-HPLC to afford Example 13 (39 mg, 7.4% yield) as awhite solid. Partial ¹H NMR (CDCl₃, 300 MHz): δ6.76 (s, 1H), 4.87 (d,J=0.6 Hz, 1H), 4.77-4.70 (m, 1H), 4.44-4.40 (m, 2H), 4.28-4.22 (m, 1H),3.34-3.26 (m, 2H), 3.17-3.08 (m, 5H), 2.83-2.74 (m, 1H), 2.38-2.30 (m,1H), 2.23 (s, 6H), 1.02 (t, J=7.2 Hz, 3H), 0.82 (t, J=7.5 Hz, 3H).LC-MS: m/z 863.8 [M+H]⁺.

Example 14:(1R,11R,13S,15S,16R,20R,21S,25S,29S)-6-chloro-21-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-25-ethyl-20-methyl-13-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-26-oxa-3,9-diazahexacyclo[14.13.0.0^(2,10).0^(4,9).0^(11,15).0^(18,29)]nonacosa-2(10),3,5,7,17-pentaene-19,27-dione

A mixture of intermediate 1 (2.0 g, 2.42 mmol) and4-Chloro-pyridin-2-ylamine (629.2 mg, 4.84 mmol) in t-BuOH (3 mL) washeated by microwave at 85° C. for 3 h. To the reaction solution wasadded water (150 mL), and the mixture extracted with dichloromethane (30mL×3). The combined organic layer was dried over sodium sulfate,filtered and concentrated. The residue was purified by chromatographyover silica gel (ethyl acetate/petroleum ether=1/1, thendichloromethane/methanol=10/1) to give the crude product, which wasfurther purified by chiral-prep-HPLC to afford the title compound (150mg, yield 7.2%). Partial ¹H NMR (CDCl₃, 400 MHz): δ7.91 (d, J=7.2 Hz,1H), 7.58 (s, 1H), 6.82 (s, 1H), 6.73 (dd, J=7.2 Hz, 2.0 Hz, 1H), 4.94(s, 1H), 4.82-4.73 (m, 1H), 4.55-4.34 (m, 3H), 2.53-2.44 (m, 1H),2.42-2.31 (m, 1H), 2.24 (s, 7H), 2.01-1.95 (m, 1H), 0.84 (t, J=7.6 Hz,3H). LCMS: m/z 856.1 [M+H]⁺.

Example 15:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(2-chlorophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-triene-16,24-dione

To a solution of Intermediate 2 (800 mg, 0.97 mmol) in ethanol (5 mL)was added 2-chlorobenzothioamide (332 mg, 1.93 mmol). The mixture wasstirred at 85° C. overnight and then heated at 120° C. under microwavefor 1 h. The mixture was concentrated to dryness and the residue waspurified by prep-HPLC and Chiral-Prep-HPLC to afford the title compound(35 mg, yield 4.0%) as a white solid. Partial ¹H NMR (CDCl₃, 400 MHz):δ8.14-8.12 (m, 1H), 7.48-7.46 (m, 1H), 7.36-7.29 (m, 2H), 6.85 (s, 1H),4.96 (s, 1H), 4.74-4.72 (m, 1H), 4.48-4.40 (m, 3H), 2.69-2.66 (m, 1H),2.24-2.20 (m, 7H), 0.85 (t, J=7.2 Hz, 3H). LCMS: m/z 901.1 [M+H]⁺.

Example 16:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-(3-methoxyphenyl)-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (800 mg, 0.96 mmol) in ethanol (20 mL)was added 3-methoxy-thiobenzamide (325 mg, 1.93 mmol). The mixture wasstirred at 85° C. overnight. The mixture was concentrated to dryness andthe residue was purified by Prep-HPLC to afford the title compound (80mg, yield 9.3%) as a white solid. Partial ¹H NMR (CDCl₃, 300 MHz):δ7.50-7.49 (m, 2H), 7.32 (t, J=10.8 Hz, 1H), 6.95-6.92 (m, 1H), 6.81 (s,1H), 4.91 (s, 1H), 4.83-4.75 (m, 1H), 4.47-4.35 (m, 3H), 3.88 (s, 3H),2.98-2.86 (m, 1H), 2.44-2.35 (m, 1H), 2.26-2.20 (m, 7H), 0.87 (t, J=7.5Hz, 3H). LCMS: m/z 895.8 [M+H]⁺.

Example 17:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-tert-butyl-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added 2,2-Dimethyl-thiopropionamide (235 mg, 2.1 mmol). The mixturewas refluxed overnight, then heated at 120° C. under microwave for 10min. The mixture was concentrated and then dissolved in ethyl acetate(60 mL), washed with saturated sodium bicarbonate aqueous (50 mL), driedover sodium sulfate, filtered and concentrated. The residue was purifiedby prep-HPLC to afford the title compound (260 mg, 28.2% yield) as awhite solid. Partial ¹H NMR (CDCl₃, 400 MHz): δ6.77 (s, 1H), 4.87 (s,1H), 4.77-4.71 (m, 1H), 4.44-4.38 (m, 2H), 4.25-4.20 (m, 1H), 3.32-3.22(m, 3H), 3.16-3.06 (m, 3H), 2.83-2.76 (m, 1H), 2.18-2.13 (m, 2H),2.03-1.83 (m, 4H), 0.83 (t, J=7.2 Hz, 3H). LCMS: m/z 844.8 [M+H]⁺.

Example 18:(1S,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-18-{[(2R,5S,6R)-6-methyl-5-(methylamino)oxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

A mixture of Example 2 (300 mg, 0.37 mmol) and sodium acetate (152 mg,1.85 mmol) in 80% methanol-H₂O (6 mL) was heated to 47° C. under N₂.Then iodine (141 mg, 0.56 mmol) was added in one portion, and the pH wasadjusted between 8-9 by addition of 1N NaOH. After 2.5 h, the reactionwas complete as monitored by LCMS. The reaction was cooled to r.t.,quenched with saturated ammonium chloride solution (10 mL), andextracted with ethyl acetate (3×10 mL). The combined organic phase waswashed with brine (10 mL), dried over anhydrous sodium sulfate, filteredand concentrated. The residue was purified by prep-HPLC to afford thetitle compound (100 mg, 34.3% yield) as a white solid. Partial ¹H NMR(400 MHz, CDCl₃): δ 7.11 (s, 1H), 4.87 (d, J=10.0 Hz, 1H), 4.72-4.67 (m,1H), 4.48-4.41 (m, 1H), 4.24 (t, J=8.8 Hz, 1H), 3.12 (t, J=7.6 Hz, 1H),2.60 (s, 3H), 2.33 (s, 3H), 0.82 (t, J=7.6 Hz, 3H); LCMS: m/z 789.8[M+H]⁺.

Example 19:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(propan-2-yl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (1.0 g, 1.21 mmol) in ethanol (15 mL)was added 2-methylpropanethioamide (187 mg, 1.82 mmol). The mixture washeated to reflux overnight, then cooled to room temperature. The mixturewas concentrated and purified by prep-HPLC to afford the title compound(230 mg, 23%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 7.14 (s,1H), 4.84 (s, 1H), 4.66-4.60 (m, 1H), 4.42 (d, J=8.8 Hz, 2H), 4.35 (q,J=6.4 Hz, 1H), 4.04 (t, J=7.6 Hz, 1H), 3.56-3.23 (m, 29H), 3.21-3.20 (m,1H), 3.10-2.94 (m, 5H), 2.81-2.74 (m, 1H), 2.36-2.33 (m, 1H), 2.19-2.04(m, 7H), 1.89 (d, J=10.8 Hz, 1H), 1.60-1.40 (m, 13H), 1.31-1.29 (m, 6H),1.21-1.06 (m, 10H), 1.08 (d, J=7.6 Hz, 3H), 0.77 (t, J=7.6 Hz, 3H).LCMS: m/z 831.4 [M+H]⁺.

Example 20:(1S,10S,12S,13R,17R,18S,22S)-22-ethyl-17-methyl-18-{[(2R,5S,6R)-6-methyl-5-(methylamino)oxan-2-yl]oxy}-5-(propan-2-yl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

Example 19 (100 mg, 0.12 mmol) and sodium acetate (49 mg, 0.60 mmol) in80% methanol-H₂O (4 mL) were heated to 47° C. under N₂. Then I₂ (46 mg,0.18 mmol) was added in one portion, and the pH was adjusted between 8-9by addition of 1N NaOH. After 2.5 h, the reaction was complete asmonitored by LCMS. The reaction was cooled to r.t., quenched withsaturated ammonium chloride solution (10 mL), and extracted with ethylacetate (3×10 mL). The combined organic phase was washed with brine (10mL), dried over anhydrous sodium sulfate, filtered and concentrated. Theresidue was purified by prep-HPLC to afford the title compound (20 mg,20.1% yield) as a white solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 7.00(s, 1H), 4.77 (d, J=1.6 Hz, 1H), 4.62-4.57 (m, 1H), 4.37-4.30 (m, 2H),4.12 (td, J₁=8.8 Hz, J₂=1.6 Hz, 1H), 2.90 (t, J=9.2 Hz, 1H), 2.73-2.66(m, 2H), 3.37-3.31 (m, 1H), 2.23 (s, 3H), 2.15-2.10 (m, 1H), 2.01-1.95(m, 1H), 1.90-1.85 (m, 1H), 1.77-1.64 (m, 2H), 1.00 (d, J=6.8 Hz, 3H),0.70 (t, J=7.6 Hz, 3H); LCMS: m/z 818.0 [M+H]⁺.

Examples 21 and 22:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(2-chlorophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(21), and(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(2-chlorophenyl)-22-ethyl-18-hydroxy-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(22)

To a solution of intermediate 1 (1.0 g, 1.2 mmol) in ethanol (15 mL) wasadded 2-Chloro-thiobenzamide (415 mg, 2.4 mmol). The mixture wasrefluxed overnight and then heated at 120° C. under microwave for 1 h.The mixture was concentrated and then dissolved in ethyl acetate (100mL), washed with saturated sodium bicarbonate aqueous (30 mL), driedover sodium sulfate, filtered and concentrated. The residue was purifiedby chromatography on silica gel column (petroleum ether/ethylacetate=2/1 then dichloromethane/methanol=30/1 to 15/1) to afford thecrude product. The crude product was further purified by prep-HPLC toafford the title compound 21 (175 mg, 16.0% yield) as a white solid andcompound 22 (39 mg, 14.1% yield) as a white solid.

Compound 21: Partial ¹H NMR (CDCl₃, 300 MHz): δ8.26 (dd, J=7.8 Hz, 1.8Hz, 1H), 7.46 (dd, J=7.5 Hz, 1.5 Hz, 1H), 7.35-7.29 (m, 2H), 6.81 (s,1H), 4.90 (s, 1H), 4.79-4.72 (m, 1H), 4.47-4.36 (m, 3H), 2.98-2.88 (m,1H), 0.84 (t, J=7.2 Hz, 3H). LCMS: m/z 899.7 [M+H]⁺.

Compound 22: Partial ¹H NMR (CDCl₃, 400 MHz): δ8.24 (dd, J=7.6 Hz, 1.6Hz, 1H), 7.46 (dd, J=8.0 Hz, 1.6 Hz, 1H), 7.35-7.29 (m, 2H), 6.83 (s,1H), 4.91 (d, J=1.2 Hz, 1H), 4.83-4.77 (m, 1H), 4.49-4.44 (m, 1H), 4.36(td, J=8.4 Hz, 1.2 Hz, 1H), 2.97-2.90 (m, 1H), 2.45-2.38 (m, 1H),2.30-2.25 (m, 1H), 0.85 (t, J=7.6 Hz, 3H). LCMS: m/z 757.8 [M+H]⁺.

Examples 23 and 24:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(3-chlorophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(22), and(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(3-chlorophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0²,¹³.0³,⁷.0⁸,¹²]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (300 mg, 0.36 mmol) in ethanol (5 mL)was added 3-Chloro-thiobenzamide (124 mg, 0.72 mmol). The mixture wasrefluxed overnight and then heated at 135° C. under microwave for 1 h.The mixture was concentrated and then dissolved in ethyl acetate (60mL), washed with saturated sodium bicarbonate aqueous (30 mL), driedover sodium sulfate, filtered and concentrated. The residue was purifiedby Prep-HPLC to afford compound Example 23 (43 mg, 15.6% yield) as awhite solid and Example 24 (160 mg, 14.7% yield) as a white solid.

Example 23: Partial ¹H NMR (CDCl₃, 400 MHz): δ7.90 (s, 1H), 7.78-7.76(m, 1H), 7.34-7.33 (m, 2H), 6.81 (s, 1H), 4.90 (d, J=0.8 Hz, 1H),4.83-4.78 (m, 1H), 4.48-4.43 (m, 1H), 4.34-4.32 (m, 1H), 2.44-2.36 (m,1H), 0.87 (t, J=7.2 Hz, 3H). LCMS: m/z 757.8 [M+H]⁺.

Example 24: Partial ¹H NMR (CDCl₃, 400 MHz): δ7.91 (s, 1H), 7.78-7.76(m, 1H), 7.35-7.32 (m, 2H), 6.79 (s, 1H), 4.90 (d, J=1.6 Hz, 1H),4.79-4.74 (m, 1H), 4.48-4.41 (m, 2H), 4.39-4.34 (m, 1H), 3.67-3.62 (m,1H), 3.29-3.11 (m, 5H), 2.94-2.86 (m, 1H), 2.42-2.35 (m, 1H), 0.86 (t,J=7.6 Hz, 3H). LCMS: m/z 898.8 [M+H]⁺.

Example 25:N-[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-5-yl]benzamide

To a solution of compound Example 1 (200 mg, 0.25 mmol) indichloromethane (6 mL) was added TEA (76 mg, 0.75 mmol) and benzoylchloride (52 mg, 0.37 mmol) at 0° C. The mixture was stirred at r.t. forovernight. The mixture was concentrated to dryness. The residue waspurified by prep-HPLC and chiral-prep-HPLC to afford the title compound(22 mg, yield 10%) as a white solid. Partial ¹H NMR (CDCl₃, 300 MHz):δ9.46-9.37 (m, 1H), 7.94 (d, J=7.5 Hz, 2H), 7.61-7.50 (m, 3H), 6.80 (s,1H), 4.90 (s, 1H), 4.77-4.73 (m, 1H), 4.49-4.42 (m, 2H), 4.21-4.15(m,1H), 2.39-2.30 (m, 1H), 2.25 (m, 7H), 0.85 (t, J=7.2 Hz, 3H). LCMS:m/z 908.8 [M+H]⁺.

Example 26:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(3-methylphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-triene-16,24-dione

To a solution of Intermediate 2 (800 mg, 0.96 mmol) in ethanol (8 mL)was added 3-methyl-thiobenzamide (290 mg, 1.94 mmol). The mixture wasstirred at 85° C. overnight. The mixture was concentrated to dryness,and the residue was purified by silica gel column chromatography(dichloromethane/methanol=20/1) then prep-HPLC to afford the titlecompound (120 mg, yield 14%) as a white solid. Partial ¹H NMR (CDCl₃,300 MHz): δ7.75 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.34-7.31 (m, 1H),7.22-7.20 (m, 1H), 6.86 (s, 1H), 4.98 (s, 1H), 4.75-4.71 (m, 1H),4.49-4.43 (m, 2H), 4.36 (t, J=8.1 Hz, 1H) 2.85-2.83 (m, 1H), 2.41 (s,3H), 2.25-2.20 (m, 7H), 0.86 (t, J=7.5 Hz, 3H). LCMS: m/z 879.8 [M+H]⁺.

Examples 27 and 28:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-[4-(trifluoromethyl)phenyl]-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(27), and(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-18-hydroxy-17-methyl-5-[4-(trifluoromethyl)phenyl]-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(28)

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added 4-trifluoromethyl-thiobenzamide (446 mg, 2.1 mmol). Themixture was refluxed overnight and then heated to 105° C. undermicrowave for 1 h. The mixture was concentrated and then dissolved inethyl acetate (60 mL), washed with saturated sodium bicarbonate (50 mL),dried over sodium sulfate, filtered and concentrated. The residue waspurified by flash column to afford Example 27 (60 mg, 5.9% yield) as awhite solid and compound Example 28 (30 mg, 31.5% yield) as a whitesolid.

Example 27: Partial ¹H NMR (CDCl₃, 400 MHz): δ8.02 (d, J=8.4 Hz, 2H),7.65 (d, J=8.4 Hz, 2H), 6.80 (s, 1H), 4.90 (d, J=1.2 Hz, 1H), 4.79-4.74(m, 1H), 4.48-4.35 (m, 3H), 2.95-2.88 (m, 1H), 2.43-2.36 (m, 1H), 0.85(t, J=7.6 Hz, 3H). LCMS: m/z 932.8 [M+H]⁺.

Example 28: Partial ¹H NMR (CDCl₃, 300 MHz): δ8.01 (d, J=8.1 Hz, 2H),7.65 (d, J=8.1 Hz, 2H), 6.81 (s, 1H), 4.90 (s 1H), 2.28-2.22 (m, 1H),0.86 (t, J=7.5 Hz, 3H). LCMS: m/z 792.3 [M+H]⁺.

Example 29:[(2S,3S,4R,5R,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-16,24-dioxo-5-(thiophen-2-yl)-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-trien-10-yl]oxy}-4,5-dimethoxy-2-methyloxan-3-yl]oxidanyl

To a solution of Intermediate 2 (1.0 g, 1.21 mmol) in ethanol (15 mL)was added 2-thiophenecarbothioamide (346 mg, 2.42 mmol). The mixture wasstirred at 85° C. overnight. The mixture was then stirred at 100° C.under microwave for 1 h. The mixture was concentrated to dryness, andthe residue was purified by silica gel chromatography(dichloromethane/methanol=20/1) and prep-HPLC to afford the titlecompound (100 mg, yield 9.5%) as a white solid. Partial ¹H NMR (CDCl₃,400 MHz): δ7.44 (dd, J₁=1.2 Hz, J₂=3.6 Hz, 1H), 7.34 (dd, J₁=0.8 Hz,J₂=4.8 Hz, 1H), 7.07-7.04 (m, 1H), 6.83 (s, 1H), 4.95 (s, 1H), 4.73-4.70(m, 1H), 4.48-4.42 (m, 2H), 4.32 (t, J=8.0 Hz, 1H),3.66-3.60 (m, 1H),2.80-2.79 (m, 1H), 2.25-2.21 (m,7H), 0.85 (t, J=7.6 Hz, 3H). LCMS: m/z870.8 [M+H]⁺.

Example 30:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-(4-methoxyphenyl)-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-triene-16,24-dione

To a solution of Intermediate 2 (450 mg, 0.54 mmol) in ethanol (3 mL)was added 4-methoxy-thiobenzamide (182 mg, 1.08 mmol). After stirring at85° C. overnight, the mixture was heated to 120° C. under microwave for1 h. The mixture was concentrated to dryness and the residue waspurified by silica gel column (dichloromethane:methanol=20:1), thenfurther purified by prep-HPLC and chiral-prep-HPLC to afford the titlecompound (12 mg, yield 2.4%) as a yellow solid. Partial ¹H NMR (CDCl₃,400 MHz): δ7.84 (d, J=8.8 Hz, 2H), 6.94 (d, J=8.8 Hz, 2H), 6.84 (s, 1H),4.94 (s, 1H), 4.73-4.70 (m, 1H), 4.55-4.53 (d, J=8.4 Hz, 1H), 4.48-4.43(m, 1H), 4.33 (t, J=8.0 Hz, 1H), 3.85 (s, 3H), 3.69-3.62 (m, 2H), 0.85(t, J=7.6 Hz, 3H). LCMS: m/z 895.8 [M+H]⁺.

Example 31:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-(4-methoxyphenyl)-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (1.0 g, 1.2 mmol) in ethanol (20 mL) wasadded 4-methoxy-thiobenzamide (405 mg, 2.4 mmol). The mixture wasstirred at 85° C. overnight. The mixture was concentrated to dryness andthe residue was purified by silica gel column(dichloromethane/methanol=20/1), then further purified by prep-HPLC toafford the title compound (60 mg, yield 5.6%) as a white solid. Partial¹H NMR (CDCl₃, 400 MHz): δ7.86-7.83 (m, 2H), 6.94-6.90 (m, 2H), 6.79 (s,1H), 4.89 (d, J=1.6 Hz, 1H), 4.78-4.75 (m, 1H), 4.45-4.41 (m, 2H), 4.34(td, J₁=8.4 Hz, J₂=1.6 Hz, 1H), 3.84 (s, 3H), 3.66-3.62 (m, 1H),3.42-3.40 (m, 1H), 3.35-3.29 (m, 2H), 3.24-3.19 (m, 1H), 3.17-3.11 (m,2H), 2.89-2.87 (m, 1H), 2.41-2.34 (m, 1H), 0.85 (t, J=7.2 Hz, 3H). LCMS:m/z 895.8 [M+H]⁺.

Examples 32 and 33:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(4-chlorophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(32), and(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(4-chlorophenyl)-22-ethyl-18-hydroxy-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione(33)

To a solution of intermediate 1 (2.0 g, 2.4 mmol) in ethanol (25 mL) wasadded 4-chloro-thiobenzamide (830 mg, 4.8 mmol). The mixture wasrefluxed overnight and then heated to 105° C. under microwave for 20min. The mixture was concentrated and then dissolved in ethyl acetate(120 mL), washed with saturated sodium bicarbonate (50 mL), dried oversodium sulfate, filtered and concentrated. The residue was purified bychromatography over silica gel (petroleum ether/ethyl acetate=1/1 thendichloromethane/methanol=30/1 to 15/1) to afford Example 32 (380 mg,17.4% yield) as a white solid and Example 33 (32 mg, 28.3% yield) as awhite solid.

Example 32: Partial ¹H NMR (CDCl₃, 400 MHz): δ7.84 (d, J=8.4 Hz, 2H),7.36 (d, J=8.8 Hz, 2H), 6.79 (s, 1H), 4.89 (d, J=1.2 Hz, 1H), 4.79-4.74(m, 1H), 4.47-4.40 (m, 2H), 4.37-4.33 (m, 1H), 2.92-2.84 (m, 1H),2.41-2.34 (m, 1H), 2.25-2.21 (m, 8H), 0.85 (t, J=7.6 Hz, 3H). LCMS: m/z899.8 [M+H]⁺.

Example 33: Partial ¹H NMR (CDCl₃, 300 MHz): δ7.84 (d, J=8.4 Hz, 2H),7.37 (d, J=8.4 Hz, 2H), 6.81 (s, 1H), 4.90 (d, J=1.2 Hz, 1H), 4.84-4.77(m, 1H), 4.49-4.42 (m, 1H), 4.33 (t, J=8.1 Hz, 1H), 3.74-3.67 (m, 1H),2.95-2.85 (m, 1H), 0.86 (t, J=7.5 Hz, 3H). LCMS: m/z 757.8 [M+H]⁺.

Example 34:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-cyclopropyl-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added cyclopropanecarbothioic acid amide (220 mg, 2.1 mmol). Themixture was refluxed overnight and then heated at 120° C. undermicrowave for 5 min. The mixture was concentrated and then dissolved inethyl acetate (60 mL), washed with saturated sodium bicarbonate (30 mL)and then brine, dried over sodium sulfate, filtered and concentrated.The residue was purified by chromatography over silica gel (petroleumether/ethyl acetate=3/1 then dichloromethane/methanol=30/1 to 10/1) toafford the crude product. The crude product was further purified byprep-HPLC to afford the title compound (360 mg, 36.9% yield) as a whitesolid. Partial ¹H NMR (CDCl₃, 400 MHz): δ6.76 (s, 1H), 4.86 (s, 1H),4.76-4.71 (m, 1H), 4.41-4.38 (m, 2H), 4.22-4.18 (m, 1H), 3.32-3.12 (m,6H), 2.81-2.73 (m, 1H), 2.36-2.29 (m, 1H), 2.16-2.11 (m, 1H), 1.98-1.95(m, 1H), 1.05-0.94 (m, 4H), 0.83 (t, J=7.2 Hz, 3H). LCMS: m/z 829.8[M+H]⁺.

Example 35:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(thiophen-2-yl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added thiophene-2-carbothioic acid amide (311 mg, 2.1 mmol). Themixture was refluxed overnight and then heated at 120° C. undermicrowave for 5 min. The mixture was concentrated and then dissolved inethyl acetate (60 mL), washed with saturated sodium bicarbonate (30 mL),dried over sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (360 mg, 38.0% yield)as a white solid. Partial ¹H NMR (CDCl₃, 400 MHz): δ7.42 (dd, J=3.6 Hz,1.2 Hz, 1H), 7.31 (dd, J=5.2 Hz, 0.8 Hz, 1H), 7.03 (dd, J=4.8 Hz, 4.0Hz, 1H), 6.77 (s, 1H), 4.88 (d, J=1.2 Hz, 1H), 4.78-4.73 (m, 1H),4.46-4.40 (m, 2H), 4.35-4.30 (m, 1H), 3.30-3.11 (m, 5H), 2.91-2.84 (m,1H), 2.40-2.34 (m, 1H), 0.85 (t, J=7.2 Hz, 3H). LCMS: m/z 870.8 [M+H]⁺.

Example 36:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(4-methylphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (500 mg, 0.61 mmol) in ethanol (6 mL)was added 4-methyl-thiobenzamide (185 mg, 1.21 mmol). The mixture wasstirred at 85° C. overnight. The mixture was concentrated to dryness,and the residue was purified by silica gel chromatography(dichloromethane/methanol=100/1-20/1), then further purified byprep-HPLC to afford the title compound (100 mg, yield 18.7%) as a whitesolid. Partial ¹H NMR (DMSO-d₆, 400 MHz): δ 7.79 (d, J=8.0 Hz, 2H),δ7.28 (d, J=8.0 Hz, 2H), 7.17 (s, 1H), 4.86 (s,1H), 4.65-4.63 (m, 1H),4.44-4.42 (m, 1H), 4.39-4.34 (m, 1H), 4.14 (t, J=8.0 Hz, 1H), 3.55-3.54(m, 2H), 3.18-2.84 (m, 6H), 0.79 (t, J=7.2 Hz, 3H). LCMS: m/z 879.8[M+H]⁺.

Example 37:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(4-methylphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-triene-16,24-dione

To a solution of Intermediate 2 (400 mg, 0.48 mmol) in ethanol (15 mL)was added 4-methyl-thiobenzamide (146 mg, 0.96 mmol). After stirring at85° C. overnight, the mixture was heated at 120° C. by microwave for 2h. The mixture was concentrated to dryness, and the residue was purifiedby prep-HPLC to afford the title compound (30 mg, yield 7.1%) as a whitesolid. Partial ¹H NMR (CDCl₃, 400 MHz): δ7.80 (d, J=8.0 Hz, 2H), 7.20(d, J=8.0 Hz, 2H), 6.79 (s, 1H), 4.90 (s, 1H), 4.78-4.75 (m, 1H),4.47-4.41 (m, 2H), 4.35 (t, J=8.4 Hz, 1H), 3.66-3.62 (m, 1H), 2.90-2.88(m, 1H), 2.37 (m, 4H), 2.25-2.20 (m, 8H), 0.85 (t, J=7.2 Hz, 3H). LCMS:m/z 879.8 [M+H]⁺.

Example 38:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(3-methylphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (800 mg, 0.96 mmol) in ethanol (6 mL)was added 3-methyl-thiobenzamide (290 mg, 1.94 mmol). The mixture wasstirred at 85° C. overnight. The mixture was concentrated to dryness,and the residue was purified by silica gel chromatography(dichloromethane/methanol=100/1-20/1), then further purified byprep-HPLC to afford the title compound (600 mg, yield 71.2%) as a yellowsolid. Partial ¹H NMR (CDCl₃, 400 MHz): δ7.73-7.69 (m, 2H), 7.30-7.26(m, 1H), 7.19-7.17 (d, J=7.2 Hz, 1H), 6.79 (s, 1H), 4.90 (d, J=1.2 Hz,1H), 4.78-4.76 (m, 1H), 4.46-4.41 (m, 2H), 4.36-4.34 (m, 1H), 3.66-3.64(m, 1H), 2.90-2.89 (m, 1H), 2.40-2.36 (m, 4H), 2.26-2.14 (m, 8H), 0.86(t, J=7.2 Hz, 3H). LCMS: m/z 878.8 [M+H]⁺.

To a solution of Spinosyn A (2.5 g, 3.42 mmol) in dichloromethane (50mL) at 0° C. was added m-CPBA (1.76 g, 10.26 mmol). The mixture wasstirred at r.t. for 3 h. 50 mL aqueous Na₂SO₃ was added into themixture. The mixture was stirred at RT for 2 h before it was extractedwith dichloromethane (50 mL×2). The organic layer was separated, driedover sodium sulfate, filtered and concentrated. The residue was purifiedby silica gel column (dichloromethane:methanol 100:1˜10:1) to afford theIntermediate 4 epoxide (2.0 g, yield 78%) as a yellow solid.

Intermediate 5:(2S,3aR,5aR,5bS,9S,13S,14R,16aR,16bS)-5-bromo-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-9-ethyl-4-hydroxy-14-methyl-2-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-1H,2H,3H,3aH,4H,5H,5aH,5bH,6H,7H,9H,10H,11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-7,15-dione

To a solution of Intermediate 4 (1.0 g, 1.33 mmol) in dichloromethane(25 mL) at 0° C. was added Triphenylphosphine-HBr (913 mg, 2.66 mmol).The mixture was stirred at r.t. for 3 h, then concentrated to dryness.The residue was purified by silica gel chromatography(dichloromethane:methanol 100:1˜20:1) to afford compound the titlecompound (1.0 g, yield 90%) as a white solid.

Example 39:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-5,22-diethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (500 mg, 0.60 mmol) in ethanol (10 mL)was added thiopropionamide (81 mg, 0.90 mmol). The mixture was stirredfor 6 h and then heated to 120° C. under microwave for 10 min. Aftercooling to r.t., the mixture was purified by flash chromatography toprovide the title compound (46 mg, 11.8%) as a white solid. Partial ¹HNMR (DMSO-d₆, 400 MHz): δ7.14 (s, 1H), 4.83 (s, 1H), 4.63-4.59 (m, 1H),4.43-4.41 (m, 1H), 4.36-4.31 (m, 1H), 4.04 (t, J=8.8 Hz, 1H), 3.53-3.28(m, 22H), 3.10-2.93 (m, 7H), 2.80-2.74 (m, 1H), 2.38-2.30 (m, 1H),2.20-2.03 (m, 8H), 1.91-1.03 (m, 28H), 0.76 (t, J=7.2 Hz, 3H). LCMS: m/z816.9 [M+H]⁺.

Example 40:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (1.0 g, 1.2 mmol) and acetamidinehydrochloride (229 mg, 2.4 mmol) in acetonitrile (15 mL) was addedpotassium carbonate (500 mg, 3.6 mmol). The mixture was heated at 85° C.for 4 h. The mixture was concentrated and purified by chromatographyover silica gel (dichloromethane/methanol=50/1 to 20/1) to afford thecrude product. The crude product was further purified by prep-HPLC toafford the title compound (210 mg, 22.1% yield) as a white solid. ¹HNMR(DMSO-d₆, 400 MHz): δ 11.45 (brs, 1H), 7.11 (s, 1H), 4.82 (s, 1H),4.65-4.59 (m, 1H), 4.41 (d, J=8.4 Hz, 1H), 4.33-4.28 (m, 1H), 3.88-3.79(m, 1H), 3.53-3.30 (m, 18H), 3.03-2.59 (m, 5H), 2.29-1.05 (m, 34H), 0.77(t, J=7.6 Hz, 3H). LCMS: m/z 785.9 [M+H]⁺.

Example 41:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(trifluoromethyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (1.0 g, 1.2 mmol) andtrifluoroacetamidine (271 mg, 2.4 mmol) in acetonitrile (15 mL) wasadded potassium carbonate (500 mg, 3.6 mmol). The mixture was heated at85° C. for 3 h. The mixture was concentrated and purified bychromatography over silica gel (dichloromethane/methanol=50/1 to 20/1)to give the crude product. The crude product was further purified byprep-HPLC to afford the title compound (120 mg, 11.8% yield) as a whitesolid. ¹H NMR (DMSO-d₆, 400 MHz): δ 13.23 (brs, 1H), 7.14 (s, 1H), 4.83(s, 1H), 4.67-4.59 (m, 1H), 4.42 (d, J=8.8 Hz, 1H), 4.37-4.30 (m, 1H),4.08-3.87 (m, 1H), 3.55-3.32 (m, 22H), 3.04-2.61 (m, 6H), 2.33-2.25 (m,1H), 2.14 (s, 6H), 2.11-1.06 (m, 19H), 0.77 (t, J=7.6 Hz, 3H). LCMS: m/z839.9 [M+H]⁺.

Example 42:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-18-hydroxy-17-methyl-5-(propan-2-yl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (2.0 g, 2.42 mmol) in ethanol (15 mL)was added 2-methylthiopropionamide (374 mg, 3.63 mmol). The mixture washeated to reflux overnight. After cooling to r.t, the mixture waspurified by prep-HPLC to afford the title compound (60 mg, 3%) as awhite solid. Partial ¹H NMR (DMSO-d₆, 400 MHz): δ 7.07 (s, 1H), 4.83 (s,1H), 4.72 (m, 1H) 4.66-4.60 (m, 1H), 4.43-4.31 (m, 2H), 4.04 (t, J=9.0Hz, 1H), 3.46-3.30 (m, 24H), 3.10-2.94 (m, 6H), 2.39-2.32 (m,1H), 2.16(s, 7H), 1.61-1.43 (m, 9H),1.31-1.29 (m, 6H), 1.17-1.16 (s, 4H),1.07-1.06 (s, 3H), 0.77 (t, J=14.4 Hz, 3H). LCMS: m/z 689.9 [M−H]⁺.

Example 43:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-sulfanylidene-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),14-diene-16,24-dione

(450 mg, 0.6 mmol) was dissolved in n-butanol (5 mL), and ammoniumthiocyanate (180 mg, 2.3 mmol) was added. The mixture was refluxed for30 min before it was cooled to r.t. The mixture was portioned betweenethyl acetate and water (60 mL/50 mL). The organic layer was washed withbrine (30 mL), dried over sodium sulfate, filtered and concentrated. Theresidue was purified by prep-HPLC to afford the title compound (82 mg,17.3% yield) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 11.96 (s,1H), 7.11 (s, 1H), 4.78 (s, 1H), 4.65-4.62 (m, 1H), 4.41 (d, J=8.8 Hz,1H), 4.33-4.26 (m, 1H), 3.87 (t, J=7.6 Hz, 1H), 3.56-3.53 (m, 2H),3.43-3.29 (m, 24H), 3.15-3.12 (m, 1H), 3.00-2.93 (m, 3H), 2.89-2.85 (m,1H), 2.46-2.44 (m, 1H), 2.27-2.05 (m, 10H), 1.98-1.05 (m, 27H), 0.76 (t,J=7.2 Hz, 3H). LCMS: m/z 803.8 [M+H]⁻.

Example 44:(1R,11R,13S,15S,16R,20R,21S,25S,29S)-21-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-25-ethyl-20-methyl-13-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-26-oxa-3,9-diazahexacyclo[14.13.0.0^(2,10).0^(4,9).0^(11,15).0^(18,29)]nonacosa-2(10),3,5,7,17-pentaene-19,27-dione

A mixture of Intermediate 1 (2.00 g, 2.42 mmol) and 2-aminopyridinehydrochloride (455 mg, 4.84 mmol) in t-BuOH (10 mL) was heated at 85° C.for overnight. The mixture was concentrated under reduced pressure togive an oil, which was purified by chromatography on silica gel column(dichloromethane/methanol=50/1 to 15/1) to give the crude product. Thecrude product was further purified by prep-HPLC to afford the titlecompound (160 mg, yield 8%) as a white solid. Partial ¹H NMR (DMSO-d₆,400 MHz): δ8.34 (d, J=6.4 Hz, 1H), 7.48 (d, J=9.2 Hz, 1H), 7.18-7.12 (m,2H), 6.83 (t, J=6.8 Hz, 1H), 4.94 (s, 1H), 4.68-4.62 (m, 1H), 4.44-4.38(m, 2H), 4.12 (t, J=8.8 Hz, 1H), 3.58-3.29 (m, 22H), 3.17-2.91 (m, 7H),2.73 (q, J=6.8 Hz, 1H), 2.38-2.31 (m, 1H), 2.15 (s, 6H), 2.08-1.08 (m,31H), 0.78 (t, J=7.4 Hz, 3H). LCMS: m/z 822.3 [M+H]⁺.

Example 45:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

A mixture of formamide (653 mg, 14.5 mmol) and P₂S₅ (537 mg, 2.4 mmol)in 10 mL of dioxane was refluxed for 2 h. To this solution was added asolution of Intermediate 1 (1.0 g, 1.2 mmol) in 5 mL of dioxane. Themixture was refluxed for 3 h before it was concentrated under reducedpressure, then neutralized with a saturated aqueous sodiumhydrogencarbonate solution. The mixture was extracted with ethyl acetate(30 mL×3). The combined organic layer was washed with brine, dried oversodium sulfate, filtered and concentrated. The residue was purified bychromatography over silica gel, eluting with dichloromethane:methanol(50:1 to 15:1) to give a yellow solid, which was then further purifiedby Prep-HPLC to afford the title compound (220 mg, 23.0% yield) as awhite solid. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.90 (s, 1H), 7.16 (s, 1H),4.85 (s, 1H), 4.66-4.60 (m, 1H), 4.48-4.45 (m, 1H), 4.38-4.33 (m, 1H),4.13 (t, J=8.0 Hz, 1H), 3.50-3.32 (m, 18H), 3.15-3.10 (m, 2H), 3.04-3.03(m, 2H), 2.96 (t, J=9.2 Hz, 1H), 2.86-2.79 (m, 1H), 2.39-2.36 (m, 2H),2.30-1.07 (m, 30H), 0.77 (t, J=7.6 Hz, 3H). LCMS: m/z 789.8 [M+H]⁺.

Intermediate 3 and Example 46:(2S,3aR,5aR,5bS,9S,13S,14R,16aR,16bS)-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-9-ethyl-14-methyl-4-(methylamino)-2-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-1H,2H,3H,3aH,4H,5H,5aH,5bH,6H,7H,9H,10H,11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-5,7,15-trione(Intermediate 3), and(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-6,17-dimethyl-5-sulfanylidene-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),14-diene-16,24-dione(46)

To a solution of intermediate 1 (900 mg, 1.0 mmol) and methylaminehydrochloride (147 mg, 2.1 mmol) in acetonitrile (15 mL) was addedpotassium carbonate (600 mg, 4.3 mmol). The mixture was then heated at85° C. for 1 h. The mixture was concentrated and the residue wasdissolved in ethyl acetate (60 mL), washed with water and brine, driedover sodium sulfate, filtered and concentrated to give intermediate 3(800 mg) as a yellow solid, which was used for next step without furtherpurification.

Intermediate 3 was dissolved in n-butanol (10 mL), and ammoniumthiocyanate (313 mg, 4.1 mmol) was added. The mixture was refluxed for30 min. The mixture was portioned between ethyl acetate and saturatedsodium bicarbonate (60 mL/50 mL). The organic layer was washed withbrine (30 mL), dried over sodium sulfate, filtered and concentrated. Theresidue was purified by prep-HPLC to afford Example 46 (40 mg, 4.7%yield) as a white solid. ¹H NMR (CDCl₃, 400 MHz): δ 10.49 (s, 1H), 6.74(s, 1H), 4.86 (s, 1H), 4.71-4.67 (m, 1H), 4.46-4.40 (m, 3H), 3.91 (t,J=8.8 Hz, 1H), 3.57-3.42 (m, 17H), 3.27-3.20 (m, 3H), 3.12 (t, J=9.2 Hz,1H), 2.97-2.92 (m, 1H), 2.66-2.63 (m, 2H), 2.52-2.38 (m, 8H), 2.29-2.25(m, 3H), 2.02-2.00 (m, 3H), 1.76-1.17 (m, 24H), 0.83 (t, J=7.6 Hz, 3H).LCMS: m/z 818.9 [M+H]⁺.

Example 47:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-amino-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-triene-16,24-dione

To a solution of Intermediate 2 (50 mg, 0.06 mmol) in DMF (2 mL) wasadded thiourea (9.2 mg, 0.12 mmol). The mixture was stirred at 80° C.under microwave for 2 h. The mixture was directly purified by Prep-HPLCto afford the title compound (8.5 mg, yield 17%) as a white solid. ¹HNMR (DMSO-d₆, 400 MHz): δ7.15 (s, 1H), 6.77 (s, 2H), 4.82 (s, 1H),4.58-4.56 (m, 1H), 4.42 (d, J=8.8 Hz, 1H), 4.29-4.27 (m, 1H), 3.82 (t,J=8.8 Hz, 1H), 3.67-3.27 (m, 17H), 3.07-2.93 (m, 3H), 2.41-2.23 (m, 2H),2.14 (s, 6H), 2.12-2.03 (m, 2H), 1.91-1.88 (m, 2H), 1.76-1.71 (m, 2H),1.56-1.11 (m, 21H), 0.76 (t, J=7.2 Hz, 3H). LCMS: m/z 804.3 [M+H]⁺.

Example 48:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4-thia-6-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),5,14-triene-16,24-dione

To a solution of Intermediate 2 (250 mg, 0.3 mmol) in ethanol (3 mL) wasadded thioacetamide (45 mg, 0.6 mmol). The mixture was stirred at refluxovernight, then the mixture was stirred at 120° C. under microwave for 2h. The mixture was concentrated to dryness and the residue was purifiedby Prep-HPLC to afford the title compound (30 mg, yield 12%) as a whitesolid. Partial ¹H NMR (DMSO-d₆, 400 MHz): δ7.18 (s, 1H), 4.86 (s, 1H),4.61-4.59 (m, 1H), 4.43 (d, J=8.8 Hz, 1H), 4.33-4.31 (m, 1H), 4.04 (t,J=8.0 Hz, 1H), 3.53-3.31 (m, 16H), 3.29-2.94 (m, 4H), 2.71-2.60 (m, 4H),2.33-1.10 (m, 38H), 0.83 (t, J=7.8 Hz, 3H). LCMS: m/z 803.3 [M+H]⁺.

Example 49:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(pyridin-2-yl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (1.0 g, 1.21 mmol) andPyridine-2-carboxamidine hydrochloride (381 mg, 2.42 mmol) inacetonitrile (15 mL) was added potassium carbonate (501 mg, 3.63 mmol).The mixture was then heated to 85° C. overnight. The mixture wasconcentrated under reduced pressure to give an oil, which was purifiedby column chromatography on silica gel (dichloromethane/methanol=30/1 to15/1) to give the crude product. The crude product was further purifiedby prep-HPLC to afford the title compound (28 mg, yield 2.7%) as a whitesolid. Partial ¹H NMR (400 MHz, CDCl₃): δ 8.48-8.45 (m, 1H), 8.16-8.10(m, 1H), 7.74-7.70 (m, 1H), 7.21-7.16 (m, 1H), 6.85 (s, 1H), 4.95 (s,1H), 4.77-4.70 (m, 1H), 4.48-4.42 (m, 2H), 4.18-4.09 (m, 1H), 3.41-3.36(m, 1H), 3.32-3.22 (m, 3H), 3.16-3.05 (m, 2H), 2.82-2.74 (m, 1H), 2.23(s, 6H). LCMS: m/z 849.1 [M+H]⁺.

Example 50:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(4-methylphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (800 mg, 0.96 mmol) and4-methyl-benzamidine hydrochloride (330 mg, 1.9 mmol) in acetonitrile(15 mL) was added potassium carbonate (400 mg, 2.9 mmol). The mixturewas then heated to 85° C. overnight. The mixture was concentrated underreduced pressure to give an oil, which was purified by chromatographyover silica gel (dichloromethane/methanol=50/1 to 15/1) to give thecrude product. The crude product was further purified by prep-HPLC toafford the title compound (105 mg, yield 12.6%) as a white solid.Partial ¹H NMR (400 MHz, CDCl₃): δ 7.70 (d, J=8.0 Hz, 2H), 7.19 (d,J=8.0 Hz, 2H), 6.71 (s, 1H), 4.91 (s, 1H), 4.80-4.74 (m, 1H), 4.43-4.39(m, 2H), 3.98-3.92 (m, 1H), 3.65-3.61 (m, 1H), 3.37-3.33 (m, 1H),3.26-3.22 (m, 2H), 3.13-3.09 (m, 1H), 2.75-2.62 (m, 3H), 2.01-1.85 (m,5H), 0.85 (t, J=7.2 Hz, 3H). LCMS: m/z 862.1 [M+H]⁺.

Example 51:(1R,11R,13S,15S,16R,20R,21S,25S,29S)-21-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-25-ethyl-20-methyl-7-phenyl-13-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-26-oxa-3,9-diazahexacyclo[14.13.0.0^(2,10).0^(4,9).0^(11,15).0^(18,29)]nonacosa-2(10),3,5,7,17-pentaene-19,27-dione

To a solution of Intermediate 1 (400 mg, 0.48 mmol) in t-BuOH (10 mL)was added 5-phenyl-pyridin-2-ylamine (165 mg, 0.96 mmol). The mixturewas then heated to 90° C. overnight and allowed to cool. The mixture wasevaporated under reduced pressure and the residue was diluted with ethylacetate (60 mL), then washed with saturated sodium bicarbonate (30 mL),water (30 mL), and brine. The organic layer was dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography over silica gel (dichloromethane:methanol=30:1 to 15:1)to give a yellow solid, which was purified by prep-HPLC to afford thetitle compound (52 mg, yield 9.5%) as a white solid. Partial ¹H NMR (400MHz, CDCl₃): δ 8.12 (s, 1H), 7.64 (d, J=9.2 Hz, 1H), 7.56-7.54 (m, 2H),7.50-7.46 (m, 2H), 7.42-7.38 (m, 1H), 7.34 (d, J=9.2 Hz, 1H), 6.84 (s,1H), 4.94 (s, 1H), 4.79-4.76 (m, 1H), 4.50-4.48 (m, 1H), 4.43-4.38 (m,2H), 3.33-3.27 (m, 3H), 3.18-3.13 (m, 2H), 3.08-3.02 (m, 1H), 2.57-2.52(m, 1H), 2.40-2.34 (m, 1H), 0.84 (t, J=6.8 Hz, 3H); LCMS: m/z 898.4[M+H]⁺.

Example 52:(1R,11R,13S,15S,16R,20R,21S,25S,29S)-5-(benzyloxy)-21-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-25-ethyl-20-methyl-13-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-26-oxa-3,9-diazahexacyclo[14.13.0.0^(2,10).0^(4,9).0^(11,15).0^(18,29)]nonacosa-2(10),3,5,7,17-pentaene-19,27-dione

To a solution of Intermediate 1 (400 mg, 0.48 mmol) in t-BuOH (10 mL)was added 3-benzyloxy-pyridin-2-ylamine (192 mg, 0.96 mmol). The mixturewas then heated to 90° C. overnight. The mixture was evaporated underreduced pressure and the residue was diluted with ethyl acetate (60 mL),then washed with saturated sodium bicarbonate (30 mL), water (30 mL),and brine. The organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography onsilica gel (petroleum ether:ethyl acetate=2:1 todichloromethane:methanol=15:1) to give a yellow solid which was furtherpurified by prep-HPLC to afford the title compound (35 mg, yield 6.2%)as a white solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 7.63 (d, J=6.4 Hz,1H), 7.51-7.49 (m, 2H), 7.38-7.35 (m, 2H), 7.32-7.30 (m, 1H), 6.82 (s,1H), 6.58-6.55 (m, 1H), 6.40 (d, J=7.2 Hz, 1H), 5.35 (s, 2H), 4.93 (s,1H), 4.79-4.78 (m, 1H), 4.49-4.47 (m, 1H), 4.42-4.38 (m, 2H), 3.36-3.13(m, 6H), 3.03-2.96 (m, 1H), 2.50-2.45 (m, 1H), 0.83 (t, J=7.2 Hz, 3H).LCMS: m/z 928.1 [M+H]⁺.

Example 53:(1R,11R,13S,15S,16R,20R,21S,25S,29S)-21-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-25-ethyl-5-methoxy-20-methyl-13-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-26-oxa-3,9-diazahexacyclo[14.13.0.0^(2,10).0^(4,9).0^(11,15).0^(18,29)]nonacosa-2(10),3,5,7,17-pentaene-19,27-dione

To a solution of Intermediate 1 (500 mg, 0.60 mmol) in t-BuOH (5 mL) wasadded 3-methoxy-pyridin-2-ylamine (150 mg, 1.2 mmol). The mixture wasthen heated to 90° C. overnight. The mixture was evaporated underreduced pressure and the residue was diluted with ethyl acetate (60 mL),then washed with saturated sodium bicarbonate (30 mL), water (30 mL),and brine. The organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography oversilica gel (petroleum ether:ethyl acetate=2:1 todichloromethane:methanol=15:1) to give the crude product, which wasfurther purified by prep-HPLC to afford the title compound (39 mg, yield7.5%) as a white solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 7.64 (d,J=6.4 Hz, 1H), 6.81 (s, 1H), 6.64 (t, J=7.6 Hz, 1H), 6.39 (d, J=7.6 Hz,1H), 4.93 (s, 1H), 4.80-4.77 (m, 1H), 4.51-4.47 (m, 1H), 4.44-4.40 (m,2H), 4.00 (s, 3H), 3.35-3.21 (m, 3H), 3.17-3.13 (m, 2H), 3.04-2.96 (m,1H), 2.51-2.46 (m, 1H), 2.38-2.31 (1m, 1H), 2.23 (s, 6H), 2.02-1.95 (m,2H), 1.86-1.84 (m, 2H). LCMS: m/z 852.1 [M+H]⁺.

Example 54:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(pyridin-3-yl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-4,6-diazapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (500 mg, 0.60 mmol) andpyridine-3-carboxamidine hydrochloride (191 mg, 1.2 mmol) inacetonitrile (10 mL) was added potassium carbonate (250 mg, 1.8 mmol).The mixture was then heated to 85° C. overnight. The mixture wasconcentrated under reduced pressure to give an oil, which was purifiedby column chromatography on silica gel (dichloromethane/methanol=30/1 to15/1) to give the crude product. The crude product was further purifiedby prep-HPLC to afford the title compound (39 mg, yield 7.6%) as a whitesolid. Partial ¹H NMR (400 MHz, CDCl₃): δ 10.39 (s br, 1H), 9.04 (s,1H), 8.54 (d, J=3.2 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.33 (dd, J=8.0 Hz,4.8 Hz, 1H), 6.81 (s, 1H), 4.92 (s, 1H), 4.74-4.70 (m, 1H), 4.47-4.42(m, 2H), 4.01-3.97 (m, 1H), 3.15-3.10 (m, 1H), 2.99-2.92 (m, 1H),2.78-2.70 (m, 1H), 0.82 (t, J=6.8 Hz, 3H). LCMS: m/z 849.1 [M+H]⁺.

Example 55:(1R,15R,17S,19S,20R,24R,25S,29S,33S)-25-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-29-ethyl-24-methyl-17-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-30-oxa-3,13-diazaheptacyclo[18.13.0.0^(2,14).0^(4,13).0^(5,10).0^(15,19).0^(22,33)]tritriaconta-2(14),3,5(10),6,8,11,21-heptaene-23,31-dione

A mixture of Intermediate 1 (250 mg, 0.3 mmol) and quinolin-2-ylamine(87 mg, 0.6 mmol) in t-BuOH (2 mL) was heated by microwave at 85° C. for3 h. The mixture was purified by prep-HPLC to afford the title compound(15 mg, yield 5.7%) as a yellow solid. Partial ¹H NMR (CDCl₃, 400 MHz):δ8.63 (d, J=8.0 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H), 7.66 (d, J=8.0 Hz, 1H),7.59 (t, J=8.0 Hz, 1H), 7.52 (dt, J=6.0, 1.2 Hz, 1H), 6.99 (d, J=6.8 Hz,1H), 6.85 (s, 1H), 4.96 (s, 1H), 4.86-4.77 (m, 1H), 4.54-4.48 (m, 1H),4.45-4.36 (m, 2H), 3.44-3.38 (m, 2H), 3.36-3.26 (m, 2H), 3.21-3.12 (m,2H), 3.10-3.01 (m, 1H), 2.56-2.49 (m, 1H), 2.41-2.32 (m, 1H), 2.25 (s,7H), 0.88 (t, J=7.6 Hz, 3H). LCMS: m/z 871.9 [M+H]⁺.

Example 56:(1S,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(phenylamino)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded phenylthiourea (170 mg, 1.1 mmol) and the resulting mixture wasstirred at reflux overnight. The reaction mixture was concentrated, andthe residue was purified by prep-HPLC to afford the title compound (150mg, 17.0% yield) as a white solid. Partial ¹H NMR (400 MHz, Acetone-d₆):δ 7.54 (d, J=8.4 Hz, 2H), 7.17 (t, J=8.0 Hz, 2H), 6.99 (s, 1H), 6.81 (t,J=7.2 Hz, 2H), 4.75 (d, J=1.6 Hz, 1H), 4.63-4.58 (m, 1H), 4.36-4.30 (m,2H), 4.04 (m, 1H), 3.07-3.05 (m, 2H), 3.01-2.96 (m, 1H), 2.90 (t, J=9.2Hz, 1H), 2.69-2.62 (m, 2H), 2.36-2.27 (m, 1H), 2.08 (s, 6H), 2.06-1.97(m, 2H), 1.09-1.06 (m, 7H), 1.01 (d, J=6.8 Hz, 3H), 0.72 (t, J=7.2 Hz,3H); LCMS: m/z 881.1 [M+H]⁺.

Example 57:N-[(2R,3S,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-18-yl]oxy}-2-methyloxan-3-yl]-N,4-dimethylbenzene-1-sulfonamide

To a solution of Example 18 (250 mg, 0.31 mmol) and DIPEA (80 mg, 0.62mmol) in dichloromethane (10 mL) was added 4-methyl-benzenesulfonylchloride (89.3 mg, 0.47 mmol). The mixture was then stirred at 0° C.under N₂ for 1 h. The reaction mixture was diluted with water (20 mL)and saturated sodium bicarbonate (20 mL), then extracted withdichloromethane (3×20 mL). The combined organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by chromatography on silica gel, followed by prep-HPLC toafford the title compound (10.5 mg, 3.6% yield) as a yellow solid.Partial ¹H NMR (400 MHz, CDCl₃): δ 7.68 (d, J=8.0 Hz, 2H), 7.29 (d,J=7.6 Hz, 2H), 6.77 (s, 1H), 4.87 (s, 1H), 4.78-4.71 (m, 1H), 4.46-4.36(m, 2H), 4.27-4.21 (m, 1H), 3.36-3.24 (m, 2H), 3.18-3.06 (m, 4H),2.85-2.76 (m, 1H), 2.81 (s, 3H), 2.71 (s, 3H), 2.43 (s, 3H),2.37-2.30(m, 1H), 2.19-2.13 (m, 1H), 0.83 (t, J=7.2 Hz, 3H); LCMS: m/z 943.0[M+H]⁺.

Example 58:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-[benzyl(methyl)amino]-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Example 18 (200 mg, 0.25 mmol) and DIPEA (64.5 mg, 0.5mmol) in dichloromethane (5 mL) was added benzyl bromide (63.7 mg, 0.37mmol). The mixture was then stirred at rt overnight. The reactionmixture was diluted with water (20 mL) and extracted withdichloromethane (3×30 mL). The combined organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (30 mg, 12.0% yield)as a yellow solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 7.33-7.28 (m, 4H),7.25-7.20 (m, 1H), 4.86 (s, 1H), 4.78-4.69 (m, 1H), 4.46-4.38 (m, 2H),4.28-4.22 (m, 1H), 3.36-3.25 (m, 2H), 3.19-3.06 (m, 4H), 2.85-2.75 (m,1H), 2.81 (s, 3H), 2.38-2.29 (m, 2H), 2.19-2.12 (m, 4H), 2.01-1.89 (m,2H), 0.83 (t, J=7.2 Hz, 3H); LCMS: m/z 880.1 [M+H]⁺.

Example 59:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-18-{[(2R,5S,6R)-6-methyl-5-[methyl(propyl)amino]oxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of propionaldehyde (43.5 mg, 0.75 mmol) and one drop ofHOAc in methanol was, added Example 18 (200 mg, 0.25 mmol). The mixturewas stirred at r.t. for 1 h. Then sodium cyanoborohydride (47.1 mg, 0.75mmol) was added and the mixture was stirred at r.t. overnight. Themixture was diluted with water (30 mL) and extracted withdichloromethane (3×30 mL). The combined organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (17.3 mg, 8.3% yield)as a yellow solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 6.78 (s, 1H), 4.88(s, 1H), 4.78-4.71 (m, 1H), 4.45-4.38 (m, 2H), 4.28-4.22 (m, 1H),3.36-3.26 (m, 2H), 3.21-3.06 (m, 4H), 2.84-2.76 (m, 1H), 2.65 (s, 3H);LCMS: m/z 831.1 [M+H]⁺.

Example 60:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-18-{[(2R,5S,6R)-6-methyl-5-{methyl[(5-methylfuran-2-yl)methyl]amino}oxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of propionaldehyde (82.6 mg, 0.75 mmol) and one drop ofHOAc in methanol was added Example 18 (200 mg, 0.25 mmol). The mixturewas stirred at rt for 1 h. At this point sodium cyanoborohydride (47.1mg, 0.75 mmol) was added, the mixture was stirred at rt overnight. Themixture was concentrated and was diluted with water (30 mL) andextracted with dichloromethane (3×30 mL). The combined organic phase wasdried over anhydrous sodium sulfate, filtered and concentrated. Theresidue was purified by prep-HPLC to afford the title compound (30.0 mg,13.6% yield) as a white solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 6.76(s, 1H), 6.02 (d, J=3.2 Hz, 1H), 5.87-5.85 (m, 1H), 4.88 (d, J=1.6 Hz,1H), 4.78-4.71 (m, 1H), 4.46-4.38 (m, 1H), 4.28-4.21 (m, 1H), 3.36-3.25(m, 2H), 3.21-3.06 (m, 4H), 2.84-2.76 (m, 1H), 2.65 (s, 3H), 2.39-2.30(m, 2H), 2.27-2.24 (m, 6H), 2.19-2.13 (m, 1H), 0.83 (t, J=7.2 Hz, 3H);LCMS: m/z 831.1 [M+H]⁺.

Example 61:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-{[(2-chloro-4-fluorophenyl)methyl](methyl)amino}-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of 2-chloro-4-fluoro-benzaldehyde (71.6 mg, 1.14 mmol) andone drop of HOAc in methanol was added Example 18 (300 mg, 0.38 mmol).The mixture was stirred at rt for 1 h. At this point sodiumcyanoborohydride (71.6 mg, 1.14 mmol) was added and the mixture wasstirred at r.t. overnight. The reaction was diluted with water (30 mL)and extracted with dichloromethane (3×30 mL). The combined organic phasewas dried over anhydrous sodium sulfate, filtered and concentrated. Theresidue was purified by prep-HPLC to afford the title compound (10.8 mg,3.0% yield) as a white solid. Partial ¹H NMR (400 MHz, CDCl₃): δ7.44-7.40 (m, 1H), 7.10-7.07 (m, 1H), 6.98-6.91 (m, 1H), 6.78 (s, 1H),4.88-4.71 (m, 2H), 4.47-4.39 (m, 2H), 4.28-4.22 (m, 1H), 3.37-3.25 (m,2H), 3.20-3.06 (m, 4H), 2.85-2.76 (m, 1H), 2.65 (s, 3H), 2.39-2.28 (m,1H), 2.03-1.92 (m, 2H), 0.83 (t, J=7.6 Hz, 3H); LCMS: m/z 931.1 [M+H]⁺.

Example 62:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-18-{[(2R,5S,6R)-5-[ethyl(methyl)amino]-6-methyloxan-2-yl]oxy}-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of acetaldehyde (50 mg, 1.14 mmol) and two drops of HOAcin methanol was added Example 18 (300 mg, 0.38 mmol). The mixture wasstirred at rt for 7 h. At this point sodium cyanoborohydride (71.6 mg,1.14 mmol) was added and the mixture was stirred at r.t overnight. Themixture was diluted with water (30 mL) and extracted withdichloromethane (3×30 mL). The combined organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (16.4 mg, 5.2% yield)as a yellow solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 6.78 (s, 1H), 4.88(s, 1H), 4.78-4.70 (m, 1H), 4.51-4.39 (m, 2H), 4.27-4.20 (m, 1H),3.37-3.31 (m, 2H), 3.21-3.06 (m, 4H), 2.85-2.76 (m, 1H), 2.65 (s, 3H),2.39-2.30 (m, 1H), 2.20-2.13 (m, 1H), 0.83 (t, J=7.2 Hz, 3H); LCMS: m/z817.1 [M+H]⁺.

Example 63:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-[cyclopropyl(methyl)amino]-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Example 18 (150 mg, 0.19 mmol) and two drops of HOAc inmethanol were added (1-ethoxy-cyclopropoxy)-trimethyl-silane (66.5 mg,0.38 mmol) and sodium cyanoborohydride (24 mg, 0.38 mmol). The mixturewas heated at reflux overnight. The mixture was then concentrated andthe residue was purified by prep-HPLC to afford the title compound (35mg, 22.2% yield) as a white solid. Partial ¹H NMR (400 MHz, CDCl₃): δ6.78 (s, 1H), 4.88 (s, 1H), 4.78-4.70 (m, 1H), 4.48-4.37 (m, 2H),4.29-4.22 (m, 1H), 3.38-3.25 (m, 2H), 3.21-3.05 (m, 4H), 2.85-2.76 (m,1H), 2.65 (s, 3H), 2.45-2.29 (m, 2H), 2.24 (s, 3H), 2.19-2.12 (m, 1H),0.83 (t, J=7.2 Hz, 3H), 0.46-0.24 (m, 4H); LCMS: m/z 829.1 [M+H]⁺.

Example 64:N-[(2R,3S,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-18-yl]oxy}-2-methyloxan-3-yl]-N-methyloctanamide

To a solution of Example 18 (250 mg, 0.31 mol) and DIPEA (75.6 mg, 0.46mmol) in dichloromethane (10 mL) was added octanoyl chloride (80.0 mg,0.62 mmol). The mixture was then stirred at 0° C. for 1 h. The reactionmixture was diluted with water (20 mL) and extracted withdichloromethane (3×20 mL). The combined organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (20 mg, 7.0% yield)as a yellow solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 6.72 (s, 1H), 4.81(s, 1H), 4.72-4.59 (m, 1H), 4.45-4.33 (m, 2H), 4.19 (t, J=8.0 Hz, 2H),3.64-3.55 (m, 2H), 3.30-3.20 (m, 2H), 3.13-3.00 (m, 4H), 2.68 (s, 2H),2.58 (s, 3H); LCMS: m/z 915.1 [M+H]⁺.

Example 65:N-[(2R,3S,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-18-yl]oxy}-2-methyloxan-3-yl]-N,2-dimethylpropanamide

To a solution of Example 18 (250 mg, 0.31 mol) and DIPEA (80.0 mg, 0.46mmol) in dichloromethane (10 mL) was added isobutyryl chloride (49.0 mg,0.46 mmol). The mixture was then stirred at 0° C. for 20 min. Thereaction mixture was diluted with water (20 mL) and extracted withdichloromethane (3×20 mL). The combined organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by prep-HPLC to afford the title compound (15 mg, 5.6% yield)as a yellow solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 6.72 (s, 1H), 4.81(s, 1H), 4.72-4.64 (m, 1H), 4.45-4.32 (m, 2H), 4.19 (t, J=8.8 Hz, 1H),3.63-3.55 (m, 2H), 3.31-3.21 (m, 2H), 3.13-3.00 (m, 4H), 2.82-2.69 (m,5H), 2.58 (s, 3H), 2.32-2.23 (m, 1H), 2.13-2.07 (m, 1H), 0.79-0.72 (m,3H); LCMS: m/z 859.1 [M+H]⁺.

Example 66:N-[(2R,3S,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-18-yl]oxy}-2-methyloxan-3-yl]-N-methylbenzamide

To a solution of Example 18 (250 mg, 0.31 mol) and DIPEA (80.0 mg, 0.46mmol) in dichloromethane (10 mL) was added benzoyl chloride (66.9 mg,0.47 mmol). The mixture was then stirred at 0° C. for 1 h. The reactionmixture was diluted with water (30 mL) and aq sodium bicarbonate (20mL), then extracted with dichloromethane (3×20 mL). The combined organicphase was dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by prep-HPLC to afford the titlecompound (20.5 mg, 7.4% yield) as a yellow solid. Partial ¹H NMR (400MHz, CDCl₃): δ 7.41-7.26 (m, 5H), 4.88 (s, 1H), 4.80-4.67 (m, 1H),4.46-4.38 (m, 2H), 4.30-4.21 (m, 1H), 2.94 (s, 2H), 2.85-2.73 (m, 2H),2.20-2.12 (m, 1H), 0.87-0.76 (m, 3H); LCMS: m/z 893.1 [M+H]⁺.

Example 67:N-[(2R,3S,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-18-yl]oxy}-2-methyloxan-3-yl]-N-methylmethanesulfonamide

To a solution of Example 18 (250 mg, 0.31 mol) and DIPEA (80.0 mg, 0.46mmol) in dichloromethane (10 mL) was added methanesulfonyl chloride(53.8 mg, 0.47 mmol). The mixture was then stirred at 0° C. for 1 h. Thereaction mixture was diluted with water (30 mL) and aq sodiumbicarbonate (20 mL) and extracted with dichloromethane (3×20 mL). Thecombined organic phase was dried over anhydrous sodium sulfate, filteredand concentrated. The residue was purified by prep-HPLC to afford thetitle compound (15 mg, 5.6% yield) as a yellow solid. Partial ¹H NMR(400 MHz, CDCl₃): δ 6.78 (s, 1H), 4.88 (s, 1H), 4.79-4.72 (m, 1H),4.48-4.40 (m, 2H), 4.28-4.22 (m, 1H), 2.20-2.13 (m, 1H), 0.83 (t, J=7.2Hz, 3H); LCMS: m/z 867.0 [M+H]⁺.

Example 68:N-[(2R,3S,6R)-6-{[(1S,2R,8R,10S,12S,13R,17R,18S,22S)-22-ethyl-5,17-dimethyl-16,24-dioxo-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-trien-18-yl]oxy}-2-methyloxan-3-yl]-N-methylprop-2-enamide

To a solution of Example 18 (50 mg, 0.06 mmol) and DIPEA (15.5 mg, 0.12mmol) in dichloromethane (5 mL) was added acryloyl chloride (8.5 mg,0.09 mmol). The mixture was then stirred at 0° C. under N₂ for 30 min.The reaction mixture was diluted with water (20 mL) and aq sodiumbicarbonate (20 mL), then extracted with dichloromethane (3×20 mL). Thecombined organic phase was dried over anhydrous sodium sulfate, filteredand concentrated. The residue was purified by chromatography over silicagel and prep-HPLC to afford the title compound (30 mg, 60% yield) as ayellow solid. Partial ¹H NMR (400 MHz, CDCl₃): δ 6.79 (s, 1H), 6.63-6.51(m, 1H), 6.38-6.24 (m, 1H), 5.76-5.64 (m, 1H), 4.88 (s, 1H), 4.80-4.70(m, 1H), 4.54-4.38 (m, 2H), 4.26 (t, J=8.0 Hz, 1H), 2.92 (s, 1H), 2.65(s, 3H), 2.39-2.30 (m, 1H), 2.20-2.13 (m, 1H), 0.83 (t, J=7.2 Hz, 3H);LCMS: m/z 843.1 [M+H]⁺.

Example 69:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-[1-(4-fluorophenyl)cyclopropyl]-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (500 mg, 0.60 mmol) in ethanol (10 mL)was added 1-(4-fluorophenyl)-cyclopropanecarbothioic acid amide (236 mg,1.2 mmol). The mixture was refluxed overnight and then concentrated. Theresidue was purified by prep-HPLC to afford the title compound (66 mg,11.8% yield) as a white solid. Partial ¹H NMR (CDCl₃, 400 MHz):δ7.45-7.42 (m, 2H), 7.03 (t, J=8.8 Hz, 2H), 6.76 (s, 1H), 4.80 (s, 1H),4.77-4.71 (m, 1H), 4.42-4.39 (m, 1H), 4.37-4.34 (m, 1H), 4.22-4.17 (m,1H), 3.66-3.61 (m, 1H), 3.31-3.27 (m, 2H), 3.16-3.15 (m, 2H), 2.12-2.03(m, 2H), 2.75-2.68 (m, 1H), 2.34-2.29 (m, 1H), 2.23 (s, 6H), 0.84 (t,J=7.6 Hz, 3H). LCMS: m/z 923.1 [M+H]⁺.

Example 70:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-(4-propoxyphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Example 11 (150 mg, 0.17 mmol) and 1-bromo-propane (63mg, 0.51 mmol) in DMF (3 mL) was added potassium carbonate (70 mg, 0.51mmol). The mixture was then stirred at reflux overnight. The reactionmixture was diluted with ethyl acetate (60 mL), washed with water (30mL×2) and brine. The organic phase was dried over anhydrous sodiumsulfate, filtered and concentrated. The residue was purified byprep-HPLC to afford the title compound (39 mg, 24.8% yield) as a whitesolid. Partial ¹H NMR (400 MHz, CDCl₃): δ 7.82 (d, J=8.8 Hz, 2H), 6.90(d, J=8.8 Hz, 2H), 6.79 (s, 1H), 4.89 (s, 1H), 4.78-4.73 (m, 1H),4.46-4.41 (m, 2H), 4.34-4.30 (m, 1H), 3.95 (t, J=6.4 Hz, 2H), 3.66-3.64(m, 1H), 3.20-3.13 (m, 2H), 2.91-2.83 (m, 1H), 2.40-2.33 (m, 1H), 2.27(s, 6H), 1.04 (t, J=7.2 Hz, 3H), 0.85 (t, J=7.6 Hz, 3H). LCMS: m/z 923.1[M+H]⁺.

Example 71:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-(4-bromophenyl)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (900 mg, 1.0 mmol) in ethanol (10 mL)was added 4-bromo-thiobenzamide (470 mg, 2.1 mmol). The mixture wasrefluxed overnight and then heated at 120° C. under microwave for 1 h.After concentration, the residue was dissolved in ethyl acetate (100 mL)and washed with saturated sodium bicarbonate aqueous (50 mL). Thecombined organic layer was dried over anhydrous sodium sulfate, filteredand concentrated. The residue was purified by chromatography over silicagel (dichloromethane/methanol=30/1 to 15/1) to afford the crude product,which was further purified by prep-HPLC to afford the title compound(130 mg, 12.6% yield) as a white solid. Partial ¹H NMR (CDCl₃, 300 MHz):δ7.77 (d, J=8.4 Hz, 2H), 7.52 (d, J=8.7 Hz, 2H), 6.79 (s, 1H), 4.89 (d,J=0.6 Hz, 1H), 4.78-4.73 (m, 1H), 4.48-4.32 (m, 3H), 2.93-2.84 (m, 1H),2.40-2.34 (m, 1H), 0.85 (t, J=7.5 Hz, 3H). LCMS: m/z 943.9, 945.9[M+H]⁺.

Example 72:(1S,10S,12S,13R,17R,18S,22S)-22-ethyl-17-methyl-18-{[(2R,5S,6R)-6-methyl-5-(methylamino)oxan-2-yl]oxy}-5-(3-methylphenyl)-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

A mixture of Example 38 (100 mg, 0.11 mmol) and sodium acetate (82 mg,0.23 mmol) in methanol/H₂O (10 mL/5 mL) was heat to 50° C. At this pointiodine (86 mg, 0.34 mmol) was added in one portion, and the pH wasmaintained between 8-9 by 1 N NaOH. After 2.5 h, the reaction wascomplete as monitored by LCMS. The reaction was cooled to r.t. and wasquenched with saturated ammonium chloride solution (10 mL) and extractedwith ethyl acetate (3×10 mL). The combined organic phase was washed withbrine (10 mL), dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by prep-HPLC to afford the titlecompound (30 mg, 30.6% yield) as a white solid. Partial ¹H NMR (400 MHz,CDCl₃): 7.73-7.69 (m, 2H), 7.29 (d, J=7.2 Hz, 1H), 7.18 (d, J=7.6 Hz,1H), 6.80 (s, 1H), 4.90 (s, 1H), 4.80-4.67 (m, 1H), 4.51-4.47 (m,2H),4.35 (t, J=7.2 Hz, 1H), 2.93-2.86 (m, 1H), 2.43-2.36 (m, 8H),2.26-2.11 (m, 5H), 0.86 (t, J=7.6 Hz, 3H); LCMS: m/z 789.8 [M+H]⁺.

Example 73:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-tert-butyl-22-ethyl-17-methyl-18-{[(2R,5S,6R)-6-methyl-5-(methylamino)oxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

A mixture of Example 17 (150 mg, 0.17 mmol) and sodium acetate (73 mg,0.88 mmol) in methanol/H₂O (5 mL/1 mL) was heated to 50° C. under N₂. Atthis point iodine (68 mg, 0.26 mmol) was added in one portion and the pHwas adjusted between 8-9 by addition of 1 N NaOH. After 2.5 h, thereaction was complete as monitored by LC-MS. The reaction was cooled tor.t. and was quenched with saturated ammonium chloride solution (10 mL)and extracted with ethyl acetate (3×10 mL). The combined organic phasewas washed with brine (10 mL), dried over anhydrous sodium sulfate,filtered and concentrated. The residue was purified by prep-HPLC toafford the title compound (60 mg, 40.8% yield) as a white solid. Partial¹H NMR (400 MHz, CDCl₃): δ 6.77 (s, 1H), 4.88 (s, 1H), 4.77-4.72 (m,1H), 4.53 (d, J=9.6 Hz, 1H), 4.44-4.39 (m, 1H), 4.26-4.21 (m, 1H),3.33-3.24 (m, 3H), 3.16-3.10 (m, 3H), 2.84-2.76 (m, 1H), 2.61 (s, 3H),2.37-2.14 (m, 4H), 2.01-1.98 (m, 2H), 0.84 (t, J=7.6 Hz, 3H); LCMS: m/z831.1 [M+H]⁺.

Example 74:(1S,10S,12S,13R,17R,18S,22S)-5-(benzylamino)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded benzylthiourea (183 mg, 1.1 mmol), and the resulting mixture wasstirred at reflux overnight. The reaction mixture was concentrated, andthe residue was purified by prep-HPLC to afford the title compound (200mg, 22.4% yield) as a white solid. Partial ¹H NMR (400 MHz, Acetone-d₆):δ 7.43 (d, J=7.2 Hz, 2H), 7.32 (t, J=7.2 Hz, 2H), 7.25 (t, J=7.2 Hz,2H), 7.08 (s, 1H), 6.97 (t, J=5.6 Hz, 1H), 4.86 (d, J=1.6 Hz, 1H),4.74-4.70 (m, 1H), 4.53-4.51 (m, 2H), 4.48-4.45 (m, 1H), 4.44-4.39 (m,1H), 4.07-4.02 (m, 1H), 2.44-2.37 (m, 1H), 2.21 (s, 6H), 0.83 (t, J=7.6Hz, 3H); LCMS: m/z 894.1 [M+H]⁺.

Example 75:(1S,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-[(2-methoxyphenyl)amino]-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (2-methoxyphenyl)-thiourea (200 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (210 mg, 23.1% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 8.55 (s, 1H), 8.39-8.37 (m, 1H), 7.12 (s, 1H),7.00-6.98 (m, 1H), 6.94-6.92 (m, 2H), 4.89 (d, J=1.2 Hz, 1H), 4.77-4.71(m, 1H), 4.49-4.43 (m, 2H), 4.18 (td, J=8.8, 1.6 Hz, 1H), 3.88 (s, 3H),3.04 (t, J=9.2 Hz, 1H), 2.49-2.42 (m, 1H), 2.21 (s, 6H), 1.22-1.19 (m,6H), 1.14 (d, J=6.8 Hz, 3H), 0.85 (t, J=7.6 Hz, 3H); LCMS: m/z 910.1[M+H]⁺.

Example 76:(1S,10S,12S,13R,17R,18S,22S)-5-[(3-chlorophenyl)amino]-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (3-chlorophenyl)-thiourea (205 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (220 mg, 24.1% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 9.36 (s, 1H), 8.18 (t, J=2.0 Hz, 1H), 7.39 (dd,J=7.6, 1.6 Hz, 1H), 7.28 (t, J=8.0 Hz, 1H), 7.13 (s, 1H), 6.95 (dd,J=7.6, 1.2 Hz, 1H), 4.89 (d, J=1.2 Hz, 1H), 4.78-4.74 (m, 1H), 4.49-4.43(m, 2H), 4.22 (td, J=8.8, 1.6 Hz, 1H), 3.04 (t, J=9.2 Hz, 1H), 2.49-2.42(m, 1H), 2.22 (s, 6H), 1.22-1.19 (m, 6H), 1.14 (d, J=6.8 Hz, 3H), 0.85(t, J=7.6 Hz, 3H); LCMS: m/z 914.0 [M+H]⁺.

Example 77:(1S,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-5-[(3-methylphenyl)amino]-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded m-tolyl-thiourea (183 mg, 1.1 mmol), and the resulting mixture wasstirred at reflux overnight. The reaction mixture was concentrated, andthe residue was purified by prep-HPLC to afford the title compound (200mg, 22.4% yield) as a white solid. Partial ¹H NMR (400 MHz, Acetone-d₆):δ 9.05 (s, 1H), 7.68 (s, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.17 (t, J=8.0 Hz,1H), 7.12 (s, 1H), 6.78 (d, J=7.2 Hz, 1H), 4.89 (d, J=1.6 Hz, 1H),4.78-4.72 (m, 1H), 4.49-4.43 (m, 2H), 4.18 (td, J=8.8, 1.6 Hz, 1H), 3.04(t, J=9.2 Hz, 1H), 2.48-2.42 (m, 1H), 2.30 (s, 3H), 2.21 (s, 6H),1.22-1.19 (m, 6H), 1.14 (d, J=6.8 Hz, 3H), 0.85 (t, J=7.6 Hz, 3H); LCMS:m/z 895.1 [M+H]⁺.

Example 78:(1S,10S,12S,13R,17R,18S,22S)-5-[(2-chlorophenyl)amino]-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (2-chlorophenyl)-thiourea (205 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (220 mg, 24.1% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 8.66 (s, 1H), 8.51 (d, J=8.0 Hz, 1H), 7.43 (dd,J=8.0, 1.2 Hz, 1H), 7.33 (td, J=8.0, 1.6 Hz, 1H), 7.13 (s, 1H), 6.95(td, J=7.6, 1.2 Hz, 1H), 4.89 (d, J=1.6 Hz, 1H), 4.76-4.69 (m, 1H),4.49-4.43 (m, 2H), 4.22 (td, J=8.8, 1.6 Hz, 1H), 3.04 (t, J=9.2 Hz, 1H),2.50-2.43 (m, 1H), 2.21 (s, 6H), 1.22-1.19 (m, 6H), 1.14 (d, J=6.8 Hz,3H), 0.84 (t, J=7.6 Hz, 3H); LCMS: m/z 916.0 [M+H]⁺.

Example 79:(1S,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-5-[(3-fluorophenyl)amino]-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (3-fluorophenyl)-thiourea (187 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (190 mg, 21.2% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 9.36 (s, 1H), 7.80 (d, J=12.0 Hz, 1H),7.33-7.27 (m, 2H), 7.13 (s, 1H), 6.72-6.65 (m, 1H), 4.88 (d, J=1.2 Hz,1H), 4.78-4.72 (m, 1H), 4.49-4.43 (m, 2H), 4.20 (td, J=8.8, 1.6 Hz, 1H),3.04 (t, J=9.2 Hz, 1H), 2.49-2.43 (m, 1H), 2.21 (s, 6H), 1.22-1.19 (m,6H), 1.14 (d, J=6.4 Hz, 3H), 0.85 (t, J=7.2 Hz, 3H); LCMS: m/z 898.1[M+H]⁺.

Example 80:(1S,10S,12S,13R,17R,18S,22S)-5-[(4-chlorophenyl)amino]-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (4-chlorophenyl)-thiourea (205 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (220 mg, 24.1% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 9.24 (s, 1H), 7.72 (d, J=8.4 Hz, 2H), 7.31 (d,J=8.4 Hz, 2H), 7.12 (s, 1H), 6.95 (dd, J=7.6, 1.2 Hz, 1H), 4.88 (s, 1H),4.77-4.70 (m, 1H), 4.49-4.43 (m, 2H), 4.18 (t, J=8.8 Hz, 1H), 3.04 (t,J=9.2 Hz, 1H), 2.49-2.43 (m, 1H), 2.22 (s, 6H), 1.22-1.19 (m, 6H), 1.14(d, J=6.8 Hz, 3H), 0.85 (t, J=7.6 Hz, 3H); LCMS: m/z 914.0 [M+H]⁺.

Example 81:(1S,10S,12S,13R,17R,18S,22S)-5-{[2-(benzyloxy)phenyl]amino}-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (2-benzyloxyphenyl)-thiourea (284 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (250 mg, 25.4% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 8.60 (s, 1H), 8.36 (d, J=8.0 Hz, 1H), 7.50 (d,J=7.2 Hz, 1H), 7.39 (t, J=7.2 Hz, 2H), 7.35-7.31 (m, 1H), 7.12 (s, 1H),7.07 (d, J=7.6 Hz, 1H), 6.97-6.88 (m, 2H), 5.22 (s, 2H), 4.88 (s, 1H),4.74-4.71 (m, 1H), 4.49-4.44 (m, 2H), 4.20-4.15 (m, 1H), 3.03 (t, J=9.2Hz, 1H), 2.49-2.42 (m, 1H), 2.21 (s, 6H), 1.22-1.19 (m, 6H), 1.14 (d,J=6.8 Hz, 3H), 0.84 (t, J=7.6 Hz, 3H); LCMS: m/z 988.1 [M+H]⁺.

Example 82:(1S,10S,12S,13R,17R,18S,22S)-5-{[3-(benzyloxy)phenyl]amino}-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-10-{[(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]oxy}-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 1 (825 mg, 1.0 mol) in ethanol (20 mL) wasadded (3-benzyloxyphenyl)-thiourea (284 mg, 1.1 mmol), and the resultingmixture was stirred at reflux overnight. The reaction mixture wasconcentrated, and the residue was purified by prep-HPLC to afford thetitle compound (240 mg, 24.3% yield) as a white solid. Partial ¹H NMR(400 MHz, Acetone-d₆): δ 9.13 (s, 1H), 7.76 (s, 1H), 7.50 (d, J=7.6 Hz,2H), 7.40 (t, J=7.2 Hz, 2H), 7.34-7.30 (m, 1H), 7.18 (t, J=8.0 Hz, 1H),7.13 (s, 1H), 6.98 (d, J=16.4 Hz, 1H), 6.61 (d, J=8.4 Hz, 1H), 5.11 (s,2H), 4.88 (s, 1H), 4.74-4.71 (m, 1H), 4.49-4.44 (m, 2H), 4.20-4.15 (m,1H), 3.04 (t, J=9.2 Hz, 1H), 2.49-2.42 (m, 1H), 2.21 (s, 6H), 1.22-1.19(m, 6H), 1.14 (d, J=6.8 Hz, 3H), 0.74 (t, J=7.6 Hz, 3H); LCMS: m/z 988.0[M+H]⁺.

Intermediate 6:(2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-2-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-9-ethyl-14-methyl-1H,2H,3H,3aH,5aH,5bH,6H,7H,9H,10H,11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-7,15-dione

To a solution of Spinosyn J (850 mg, 0.85 mmol) in ethyl bromide (15 mL)was added a mixture of powered potassium hydroxide/tetra-n-butylammonium iodide (10:1, 1.0 g/0.1 g). The mixture was stirred at rt for 2h, then evaporated under reduced pressure. The residue was diluted withwater (60 mL) and extracted with ethyl acetate (50 mL×2). The combinedorganic layer was washed with brine, dried over sodium sulfate, filteredand concentrated in vacuo to give an oil, which was purified bychromatography over silica gel (dichloromethane:methanol=50:1 to 20:1)to provide the title compound (690 mg, 78.1%) as a white solid. LCMS:m/z 746.0 [M+H]⁺.

Intermediate 7:(2S,3aR,5aR,5bS,9S,13S,14R,16aR,16bS)-4-bromo-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-2-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-9-ethyl-5-hydroxy-14-methyl-1H,2H,3H,3aH,4H,5H,5aH,5bH,6H,7H,9H,10H,11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-7,15-dione

To a solution of Intermediate 6 (640 mg, 0.85 mmol) in DMSO (6 mL) wasadded dropwise a solution of concentrated sulfuric acid (84 mg, 0.85mmol) in water (2 mL). The mixture was then cooled to 0° C., and NBS(181 mg, 1.0 mmol) was added. After stirring for 30 min at 0° C., ethylacetate (100 mL) and saturated aqueous sodium bicarbonate (30 mL) wereadded. The organic layer was washed with water and brine, dried oversodium sulfate, filtered and concentrated in vacuo to give the titlecompound (750 mg, 96.2%) as a white solid, which was used for next stepwithout further purification. LCMS: m/z 842.0, 844.0 [M+H]⁺.

Intermediate 8:(2S,3aR,5aR,5bS,9S,13S,14R,16aR,16bS)-4-bromo-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-2-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-9-ethyl-14-methyl-1H,2H,3H,3aH,4H,5H,5aH,5bH,6H,7H,9H,10H,11H,12H,13H,14H,15H,16aH,16bH-as-indaceno[3,2-d]oxacyclododecane-5,7,15-trione

Dess-Martin periodinane (377 mg, 0.89 mmol) was added portionwise to asolution of Intermediate 7 (750 mg, 0.89 mmol) in dichloromethane (20mL) at 0° C. The mixture was stirred at r.t. overnight. The mixture wasdiluted with dichloromethane (80 mL), and was then washed with saturatedsodium bicarbonate (30 mL), saturated Na₂SO₃ (20 mL), and brine. Theorganic layer was dried over sodium sulfate, filtered and concentratedin vacuo to give an oil which was purified by column chromatography oversilica gel (DCM/methanol=50/1 to 15/1) to afford the title compound (705mg, 94.2%) as a white solid. LCMS: m/z 840.1, 842.1 [M+H]⁺.

Example 83:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-5,17-dimethyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 8 (300 mg, 0.35 mmol) in ethanol (5 mL)was added thioacetamide (53 mg, 0.71 mmol). The mixture was refluxedovernight. The mixture was evaporated under reduced pressure, and theresidue was diluted with ethyl acetate (60 mL). The mixture was washedwith saturated sodium bicarbonate (20 mL), water (20 mL), and brine. Theorganic layer was dried over sodium sulfate, filtered and concentrated.The residue was purified by column chromatography over silica gel (frompetroleum ether:ethyl acetate=3:1 to dichloromethane:methanol=20:1) togive a yellow solid, which was further purified by prep-HPLC to affordthe title compound (66 mg, 22.6% yield) as a white solid. Partial ¹H NMR(CDCl₃, 400 MHz): δ6.77 (s, 1H), 4.84 (s, 1H), 4.77-4.71 (m, 1H),4.42-4.38 (m, 2H), 4.26-4.21 (m, 1H), 3.76-3.68 (m, 1H), 3.65-3.60 (m,2H), 3.35-3.27 (m, 2H), 3.19-3.18 (m, 2H), 3.14-3.06 (m, 2H), 2.83-2.75(m, 1H), 2.64 (s, 3H), 2.37-2.30 (m, 1H), 2.17-2.12 (m, 1H), 1.97-1.95(m, 1H), 1.86-1.84 (m, 1H), 1.77-1.73 (m, 1H), 0.82 (t, J=7.6 Hz, 3H).LCMS: m/z 817.1 [M+H]⁺.

Example 84:(1S,2R,8R,10S,12S,13R,17R,18S,22S)-5-cyclopropyl-18-{[(2R,5S,6R)-5-(dimethylamino)-6-methyloxan-2-yl]oxy}-10-{[(2R,3R,4R,5S,6S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl]oxy}-22-ethyl-17-methyl-23-oxa-6-thia-4-azapentacyclo[13.10.0.0^(2,13).0^(3,7).0^(8,12)]pentacosa-3(7),4,14-triene-16,24-dione

To a solution of Intermediate 8 (300 mg, 0.35 mmol) in ethanol (5 mL)was added cyclopropanecarbothioic acid amide (72 mg, 0.71 mmol). Themixture was refluxed overnight. The mixture was concentrated, and theresidue was then diluted with ethyl acetate (60 mL). The mixture waswashed with saturated sodium bicarbonate (20 mL), water (20 mL), andbrine. The organic layer was dried over sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography oversilica gel (from petroleum ether:ethyl acetate=3:1 todichloromethane:methanol=20:1) to give a yellow solid, which was furtherpurified by prep-HPLC to afford the title compound (102 mg, 34.0% yield)as a white solid. Partial ¹H NMR (CDCl₃, 400 MHz): δ6.76 (s, 1H), 4.84(d, J=1.6 Hz, 1H), 4.77-4.71 (m, 1H), 4.42-4.38 (m, 2H), 4.23-4.18 (m,1H), 3.76-3.70 (m, 1H), 3.65-3.62 (m, 2H), 3.32-3.27 (m, 2H), 3.17-3.05(m, 4H), 2.82-2.74 (m, 1H), 2.36-2.28 (m, 1H), 2.16-2.11 (m, 1H),1.06-0.94 (m, 4H), 0.83 (t, J=7.6 Hz, 3H). LCMS: m/z 843.1 [M+H]⁺.

Example 85: Testing Compounds for Insecticide, Miticide, and NematicideUtility

The compounds produced by the methods described above are tested foractivity against a number of insects, mites, and nematodes. Successfulcompounds are useful for reducing populations of insects, mites, and/ornematodes, and are used in a method of inhibiting an insect, mite,and/or nematode population after application to a locus of the pest aneffective insect-, mite-, or nematode-inactivating amount of a compound.

Activity against Spodoptera species:

Cotton leaf discs are placed on agar in 24-well microtiter plates andsprayed with serial dilutions of aqueous test solutions prepared fromDMSO stock solutions, with a highest dose of 200 ppm. After drying, theleaf discs are infested with L1 larvae and samples are assessed formortality after 4 to 8 days.

Activity against Plutella species:

Artificial diet optimized for Lepidopteran species is aliquoted into24-well microtiter plates and treated with aqueous test solutionsprepared from DMSO stock solutions by pipetting, with a highest dose of200 ppm. After drying, the plates are infested with L2 larvae andmortality is assessed after 4 to 8 days.

Activity against Diabrotica species:

Artificial diet optimized for Coleopteran species is aliquoted into24-well microtiter plates and treated with aqueous test solutionsprepared from DMSO stock solutions by pipetting, with a highest dose of200 ppm. After drying, the plates are infested with L2 larvae andmortality is assessed after 4 to 8 days.

Activity against Myzus species:

Sunflower leaf discs are placed on agar in a 24-well microtiter plateand sprayed with serial dilutions of aqueous test solutions preparedfrom DMSO stock solutions, with a highest dose of 200 ppm. After drying,the leaf discs are infested with a mixed age aphid population andsamples are assessed for mortality after 4 to 8 days.

Activity against Thrips species:

Sunflower leaf discs are placed on agar in a 24-well microtiter plateand sprayed with serial dilutions of aqueous test solutions preparedfrom DMSO stock solutions, with a highest dose of 200 ppm. After drying,the leaf discs are infested with a mixed age thrips population andsamples are assessed for mortality after 4 to 8 days.

Activity against Euschistus species:

Sunflower leaf discs are placed on agar in a 24-well microtiter plateand sprayed with serial dilutions of aqueous test solutions preparedfrom DMSO stock solutions, with a highest dose of 200 ppm. After drying,the leaf discs are infested N2 nymphs and samples are assessed formortality after 4 to 8 days.

Activity against Tetranychus species:

Bean leaf discs are placed on agar in a 24-well microtiter plate andsprayed with serial dilutions of aqueous test solutions prepared fromDMSO stock solutions, with a highest dose of 200 ppm. After drying, theleaf discs are infested with mixed mobile populations and samples areassessed for mortality after 4 to 8 days.

Activity against Meloidogyne species:

Untreated cucumber seeds are placed into the bottom of a clear cup towhich clean white sand is added. The cups are sprayed with aqueous testsolution while rotating on a pedestal allowing the test solution to bedeposited on the sand. To each cup is dispensed water containingnematodes. After 10 to 14 days the nematode populations are assessed formortality.

Activity against Blattella species:

Green insect diet material is dispensed into a diet cup onto whichaqueous test solution is sprayed. Treated cups are air dried andinfested with late third or early fourth instar cockroaches. After 10 to14 days the cockroach populations are assessed for mortality.

Activity against Aedes species:

L2 Aedes larvae in a nutrition mixture are placed in 96-well microtiterplates. Aqueous test solutions are pipetted into the wells. After 1 to 3days the mosquito populations are assessed for mortality.

All examples described above were active at test solution concentrationsof 200 ppm or below against at least one of the above test organisms.

It is understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and the terminology is notintended to be limiting. The scope of the invention will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber, which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application werespecifically and individually indicated to be incorporated by reference.Furthermore, each cited publication, patent, or patent application isincorporated herein by reference to disclose and describe the subjectmatter in connection with which the publications are cited. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the invention describedherein is not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided might be differentfrom the actual publication dates, which may need to be independentlyconfirmed.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featureswhich may be readily separated from or combined with the features of anyof the other several embodiments without departing from the scope orspirit of the invention. Any recited method may be carried out in theorder of events recited or in any other order that is logicallypossible. Although any methods and materials similar or equivalent tothose described herein may also be used in the practice or testing ofthe invention, representative illustrative methods and materials are nowdescribed.

What is claimed is:
 1. A spinosyn compound of the following formula:

or a salt thereof, wherein:

is a single bond or a double bond; A is selected from the groupconsisting of hydrogen, substituted or unsubstituted carbonyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl; B is selected from the group consisting ofsubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl; C is O or NH; R¹ is C₁-C₆ alkyl or C₁-C₆ aryl;and X¹, X², and X³ are each independently selected from O, S, N, NR, andCR, wherein each R is independently selected from hydrogen, hydroxyl,substituted or unsubstituted amino, substituted or unsubstituted thio,substituted or unsubstituted alkoxy, substituted or unsubstitutedaryloxy, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, wherein the ring including X¹,X², and X³ is aromatic; wherein when X¹ and X² are selected from NR andCR, the R groups of X¹ and X² optionally combine to form a substitutedor unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and wherein when X² and X³ areselected from NR and CR the R groups of X² and X³ optionally combine toform a substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkenyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.
 2. The spinosyn compound of claim 1, wherein X¹ and X² areselected from NR and CR, and wherein the R groups of X¹ and X² combineto form a substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkenyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.
 3. The spinosyn compound of claim 1, wherein X² and X³ areselected from NR and CR, and wherein the R groups of X² and X³ combineto form a substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkenyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heterocycloalkenyl,substituted or unsubstituted aryl, or and substituted or unsubstitutedheteroaryl.
 4. The spinosyn compound of claim 1, wherein A isforosamine; B is a [(2R,3R,4R,5S,6S)-3,4,5-trimethoxy-6-methyloxan-2-yl]group or a (2R,5S)-4-ethoxy-3,5-dimethoxy-6-methyloxan-2-yl] group; C isO; R¹ is ethyl; X¹ is N; X² is CR; R is alkyl, amino, or cycloalkyl; andX³ is S.
 5. The spinosyn compound of claim 4, having the followingformula:


6. The spinosyn compound of claim 4, having the following formula:


7. The spinosyn compound of claim 1, having the formula represented byStructure I-A:


8. The spinosyn compound of claim 1, having the formula represented byStructure I-B:


9. The spinosyn compound of claim 1, having the formula represented byStructure I-C:


10. The spinosyn compound of claim 1, having the formula represented byStructure I-D:


11. The spinosyn compound of claim 1, having the formula represented byStructure I-E:


12. The spinosyn compound of claim 1, having the formula represented byStructure I-F:


13. The spinosyn compound of claim 1, having the formula represented byStructure I-G:


14. A formulation comprising at least one spinosyn compound of claim 1and an acceptable carrier.
 15. The formulation of claim 14, furthercomprising at least one additional active ingredient.
 16. Theformulation of claim 15, wherein the additional active ingredient is acontact-acting insecticide or a contact-acting miticide.
 17. A methodfor controlling pests, comprising contacting a pest with an effectiveamount of the a spinosyn compound of claim
 1. 18. The method of claim17, wherein the pest is an insect, an arachnid, or a nematode.
 19. Amethod for making a spinosyn compound, comprising reacting the C-5,6double bond of Spinosyn A to form a spinosyn compound according to claim1, wherein the spinosyn compound forms via an α-halo ketoneintermediate.